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Regeneration of injured peripheral nerves is an extremely complex process. Nogo-A (neurite outgrowth inhibitor-A) inhibits axonal regeneration by interacting with Nogo receptor in the myelin sheath of the central nervous system (CNS). The aim of this study was to investigate the effects of Nogo-A and its receptor on the repair of sciatic nerve injury in rats. Sprague-Dawley rats (n=96) were randomly divided into 4 groups: control group (control), sciatic nerve transection group (model), immediate repair group (immediate repair), and delayed repair group (delayed repair). The rats were euthanized 1 week and 6 weeks after operation. The injured end tissues of the spinal cord and sciatic nerve were obtained. The protein expressions of Nogo-A and Nogo-66 receptor (NgR) were detected by immunohistochemistry. The protein expressions of Nogo-A, NgR, and Ras homolog family member A (RhoA) were detected by western blot. At 1 week after operation, the pathological changes in the immediate repaired group were less, and the protein expressions of Nogo-A, NgR, and RhoA in the spinal cord and sciatic nerve tissues were decreased (P<0.05) compared with the model group. After 6 weeks, the pathological changes in the immediate repair group and the delayed repair group were alleviated and the protein expressions decreased (P<0.05). The situation of the immediate repair group was better than that of the delayed repair group. Our data suggest that the expression of Nogo-A and its receptor increased after sciatic nerve injury, indicating that Nogo-A and its receptor play an inhibitory role in the repair process of sciatic nerve injury in rats.
Subject(s)
Animals , Rats , Receptors, Cell Surface , Myelin Proteins , Sciatic Nerve , Rats, Sprague-Dawley , GPI-Linked Proteins , Nogo Proteins , Nerve RegenerationABSTRACT
Objective To investigate the phased expression of gene and protein of NogoA and its receptor (NgR) that affects axon growth of spinal cord injury (SCI), and to explore the time window effect of electroacupuncture on SCI rats. Methods A total of 144 female Sprague-Dawley rats were randomly assigned to sham operation group (group A, n=48) and model group (n=96). In the model group, Allen's method was used to establish SCI rats model, and they were further subdivided into model control group (group B, n=48) and electroacupuncture group (group C, n=48). Group C received electroacupuncture on Dazhui (GV14), Yaoyangguan (GV3), bilateral Ciliao (BL32) and Zu-sanli (ST36) with loose-tight wave, for 20 minutes every day, one day, seven days and 14 days after modeling. The rats at every interventional therapy time were randomly subdivided into two subgroups, which accepted sev-en or 14 days of treatment. Groups A and B were killed and the injured spinal cord tissue was extracted one day, three days, seven days, 14 days and 28 days after modeling, group C at the corresponding time. The hind limb motor function was assessed with BBB score before all of rats were killed. Four samples at every time in each group were randomly selected to detect the expression of mRNA and protein of NogoA and NgR at different stage of SCI using reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR) and Western blotting. Results The BBB score began to increase 14 days after modeling, and significantly increased until 28 days after model-ing (P<0.05), compared with one day, three days and seven days after modeling in group B. The BBB score in-creased in group C than in group B at all the time points (P<0.05), except 14 days after electroacupuncture one day after modeling. The BBB score was higher as electroacupuncture intervening seven days and 14 days after modeling than that at one day after modeling in group C, and no significant difference was found between seven days and 14 days of treatment at either electroacupuncture time point (P>0.05). The expression of gene and pro-tein of NogoA and NgR in group B was in the increasing tendency after SCI, and was at the peak until 21 days af-ter modeling, and was higher in group B than in group A at each time point (P<0.01). The expression of gene and protein of NogoA decreased at all the time points in group C than in group B (P<0.05), except seven days of elec-troacupuncture intervening one day after modeling in the expression of NogoA mRNA (P>0.05). The expression of gene and protein of NogoA and NgR was lower as electroacupuncture intervening 14 days after modeling than one day after modeling in group C (P<0.05). There was no significant difference in the expression of gene and protein of NogoA and NgR between electroacupuncture intervening 14 days and seven days after modeling, and seven days and one day after modeling (P>0.05); as well as between seven days and 14 days of treatment at each time point (P>0.05). Conclusion Elerctroacupuncture could improve the hind limb motor function, which may associate with the inhibition of the expression of gene and protein of NogoA and NgR in injured spinal cord of rats after SCI. Elerctroacu-puncture is effective in the treatment of SCI at the early time, however, it is much better in the recovery stage.
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Objective: To investigate effects of Rhizoma Ligustici Chuanxiong and Radix Paeoniae Rubra on the relative expression of Nogo-A/NgR/RhoA/ ROCK mRNA in acute cerebral infarction rats. Methods: Healthy male SD rats were randomly divided into sham operation group, blank group, low dose, medium dose and high dose Rhizoma Ligustici Chuanxiong and Radix Paeoniae Rubra group, Ginkgo biloba group, Nimodipine group, and each group was divided into 3 days, 7 days, 14 days three time points. Real-Time quantitative polymerase chain reaction (PCR) was used to detect Nogo-A/NgR/RhoA/ROCK mRNA relative expression changes of acute cerebral infarction rats. Results: Compared with the blank group and the sham operation group, the relative expression level of Nogo-A/NgR/RhoA/ROCK mRNA was increased in the model group both at 3 days, 7 days and 14 days (P ＜ 0.05). After the treatment of Rhizoma Ligustici Chuanxiong and Radix Paeoniae Rubra, other than there was no significant difference between the low-dose group and the model group except for 7 days, the relative expression level of Nogo-A/NgR/RhoA/ROCK mRNA in Rhizoma Ligustici Chuanxiong and Radix Paeoniae Rubra groups was lower than that in the model group (P ＜ 0. 05). Conclusion: The relative expression level of Nogo-A/NgR/RhoA/ROCK mRNA in acute cerebral infarction rats can be reduced by Rhizoma Ligustici Chuanxiong and Radix Paeoniae Rubra.
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@#AIM: To investigate the effect of NgR mediated oxidative stress on the apoptosis of retinal ganglion cells(RGCs)induced by glucose. <p>METHODS: RGC-5 cell were divided into 3 groups: control group(DMEM high glucose medium+10% fetal calf serum), high glucose group(DMEM high glucose medium+10% fetal calf serum+30mmol/L glucose),NEP1-40 group(DMEM high glucose medium+10% fetal calf serum+30mmol/L glucose+1μmol/L NEP1-40). Detections were performed after 3d culture: the state of cell growth was observed by microscopy. Cell viability was detected by CCK-8 kit. The apoptosis rate of RGC cells was detected by flow cytometry(FCM). The intensity of ROS of the cells were detected by fluorescence microscopy. Intracellular MDA levels and SOD activity were measured by related kits. Western blot was used to detect the expressions of Bcl-2 and Bax proteins. <p>RESULTS: Compared with control group, high glucose group had a poor state and cell viability decreased, cell apoptosis rate significantly increased, ROS and MDA levels were significantly enhanced, SOD activity decreased, and the expression of anti-apoptotic protein Bcl-2 was decreased and the expression of pro apoptotic protein Bax was up-regulated. Compared with glucose group, after NgR expression was inhibited by NEP1-40, the oxidative stress reaction was reduced, Bcl-2/Bax was increased, the cell status was improved, the cell viability was increased, and the apoptosis rate was decreased in the NEP1-40 group(<i>P</i><0.05).<p>CONCLUSION: High concentration of glucose can induce apoptosis of RGC-5 cells by NgR mediated oxidative stress reaction.
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Objective To investigate the effect of transplantation with the three-dimensional spheroid-cultured mesenchymal stem cells (MSCs) on the expression of Nogo-A and NgR in rats with cerebral ischemia-reperfusion injury. Methods The experimental animals were randomly divided into Sham group, Vehicle group and MSCs treated group. The model of focal ischemia-reperfusion in rats was induced by intraluminal middle cerebral artery (MCA) occlusion with a nylon monofilament suture in Vehicle group and MSCs treated group. The fishing line was unpluged for reperfusion 2 h after ischemia and MSCs were transplanted in MSCs treated group one day later. Equivalent medium solution was given to the Vehicle group 1 d later. On the 1st day, 3rd day, and 7th day after transplantation, the neuromotor function of the animals was detected. The brain tissue of rats was harvested for RT-PCR detection of Nogo-A and NgR mRNA expression in the brain tissue of rats, and Western blotting analysis was used to detect the expression of Nogo-A and NgR protein. Results Compared with the Vehicle group, the neuromotor function was significantly improved in MSCs treated group on the 7th day; and the expressions of Nogo-A and NgR mRNA and protein were significantly down-regulated in MSCs treated group on the 1st day, 3rd day, and 7th day after transplantation (P<0.05). Conclusion Transplantation of the three-dimensional spheroid-cultured MSCs can improve the neuromotor function following cerebral ischemia/reperfusion injury, and its mechanism may be associated with down-regulation of Nogo-A and NgR in the brain tissue.
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OBJECTIVE: To investigate the best method for preparing NGR-SWCNTs-paclitaxel and observe its targeting efficiency. METHODS: SWCNTs-paclitaxel was prepared by solution mixing, and then conjugated with NGR to obtain a novel paclitaxel delivery system: NGR-SWCNTs-paclitaxel. Taking loading efficiency and encapsulate efficiency as index, studied the influential factors of the preparation of NGR-SWCNTs-paclitaxel by surfactant, times and frequency of probe sonography, quantity of carbon nano tube. The drug concentration in different tissue were detected by high performance liquid chromatography (HPLC). The targeting efficiency were used to evaluate the tissue targeting of NGR-SWCNTs-paclitaxel, SWCNTs-paclitaxel and paclitaxel. RESULTS: SWCNTs-paclitaxel was prepared by SWCNTs-paclitaxel was 1:2; Poloxamerl88-phenylalanine was 7:3; probe sonography 600 W, 15 times. SWCNTs-paclitaxel conjugated with NGR formed NGR-SWCNTs-paclitaxel. Its loading efficiency was (83.9 ±2.7)% and encapsulate efficiency was (69.3 ± 1.5)%. The Zeta potential was (-22.6 ± 1.5) mV, partical size was about (182. 1 ± 2.4) nm. The AUC of NGR-SWCNTs-paclitaxel and SWCNTs-paclitaxel in mice slpeen, liver, lung and tumor were increased obviously compared with paclitaxel (P < 0.05, P < 0.01). The targeting efficiency of SWCNTs-paclitaxel and NGR-SWCNTs-paclitaxel in heart and kidney were decreased (P < 0.05), and in tumor the targeting efficiency was 6.78% and 21.33% separately, the difference was significantly(P < 0.01). CONCLUSION: The preparation of NGR-SWCNTs-paclitaxel was practicable by solution mixing. The loading efficiency and encapsulate efficiency of NGR-SWCNTs-paclitaxel are higher. NGR-SWCNTs-paclitaxel can enhance tumor targeting of paclitaxel obviously.
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Objective To evaluate the effect of free NgR-modified bone marrow stromal cell (BMSC) transplantation on axon regeneration in rats after spinal cord injury. Methods Genes encoding free NgR protein were cloned and transduced into BMSCs at passage 3 using a lentivirus vector.Indirect immunofluorescence was used to detect the expression of free NgR protein.Meanwhile a spinal cord contusion model was established in 36 adult Sprague-Dawley rats at the T10 segment.The rats were then divided randomly into an experimental group and a control group.NgR + BMSCs were transplanted into the injured site 1 week post-trauma in the experimental group.BMSCs were also transplanted at the same time into the control group.Expression of free NgR at the injury site was detected by immunohistochemical staining at 1 week post-transplantation.The functional recovery of both groups was evaluated at 4 and 6 weeks post-transplantation.Longitudinal sections of the spinal cord were studied for axon regeneration using horseradish peroxidase staining. Results Expression of free NgR was found in the cell plasma of BMSCs by indirect immunofluorescence post-transfection.Positive immunohistochemical staining for NgR was found at the transplant site in the experimental group 1 week post-transplantation.Better axon plasticity could be observed in the experimental group.The Basso-Beattie-Bresnahan scoring of the experimental group was significantly higher than that of the controls at both observation times. Conclusions Free NgR-modified BMSCs can prompt injured axons to regenerate and thus to promote the recovery of neurological function.This might provide a new strategy to treat spinal cord injury.
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Objective To study the effects of rehabilitation training combined with the transplantation of bone marrow mesenchymal stem cells-derived neural stem cells (BMSC-D-NSCs) on the expression of Nogo-A and NgR protein in rats after spinal cord injury (SCI).Methods The spinal cords of eighty Sprague-Dawley rats were injured using a modified Allen′s impactor (H = 25 mm) at T10. The injured rats were randomly divided into a combination therapy group which was given rehabilitation training and cell transplants, a cell graft group, a rehabilitation training group and a control group. At the 7th day post SCI, BMSC-D-NSCs were transplanted into the injured spinal cords of the rats in the combination therapy and cell graft groups. Hindlimb movement was assessed using the BassoBeattie-Bresnahan (BBB) scale every week, and protein was extracted from the injured spinal cord tissue for Nogo-A and NgR determination by Western blotting at the 1st, 3rd and 7th day after cell transplantation.Results The average BBB score of the rats in the combination therapy group was significantly higher than that of the other groups from 2 weeks post transplantation. The scores in the rehabilitation training group were significantly higher than in the control group from the 5th week post transplantation. Western blotting showed high expression of Nogo-A and NgR protein 24 h post surgery, but these declined with time. For Nogo-A there was a significant difference among the groups at all three time points. In the combination therapy group the expression declined to a minimum by the 7th day. For NgR protein there was no significant difference between the 1st and 3rd day in any group.Conclusions Rehabilitation training combined with BMSC-D-NSC transplantation can have a synergistic effect on functional recovery from SCI. It can down regulate the expression of Nogo-A and NgR protein.
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Objective To sythesize 99Tcm labeled asparagine-glycine-arginine (NGR)- interferon (INF)-α2a and investigate its biodistribution by scintigraphy in tumor bearing mice. Methods NGR-INFα2a was labeled with 99Tcm by a two-step method. Ethylenedicysteine (EC) and MDP were used as bifunctional and transferring chelating agents. The bioactivities of 99Tcm-NGR-IFN-EC-NGR-IFN-α2a, EC-NGRIFN-α2a and NGR-IFN-α2a were compared using least significant difference t-test. The hepatoma bearing mice models were established by subcutaneous injection of MHCC97-H cells. The mice were randomly divided into eight groups and 7.4 MBq 99Tcm-NGR-IFN-α2a was injected via the tail vein. The tissue uptake of the radiolabeled compound was measured as % ID/g. The scintigraphy was performed at 0.5, 1, 2, 4,6, 8, 12 and 24 h after injection. ROI were drawn around tumor and non-tumor tissue and the radioactivity ratio of T/NT was calculated. Results Both the labeling efficiency and radiochemical purity of 99Tcm-EC-NGR-IFN-α2a were more than 90%. The radiochemical purity was 71% after 24 h in saline. The bioactivity showed no significant difference among three compounds (t = 0.416, 0. 120 and 1. 300, all P >0.05). The tracer was mainly excreted through alimentary and urinary tract within 24 h after injection. The peak values of % ID/g in kidney, liver, interstinal tract and tumor were 41.5 ± 8.0_ (at 8 h), 31.3 ± 5.0(at 6 h), 36.0 ± 7.8 (at 6 h), 43.0 ± 4.8 (at 4 h), respectively. The tracer was cleared quickly from the blood and the highest T/NT ratio was 16.5. The optimal imaging time ranged from 4 to 8 h after injection. Conclusions The sythesis of 99Tcm-NGR-IFN-α2a is applicable and it may be used as a potential tumor imaging agent.