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OBJECTIVE：To study the imp rovement effects of 2-dodecyl-6-methoxycyclohexa-2，5-diene-1，4-dione（DMDD） from Averrhoa carambola on H 9c2 myocardial cell injury induced by high glucose and its mechanism. METHODS ：H9c2 myocardial cells were divided into normal group ，high glucose group ，osmotic pressure control group ，DMDD high ，medium and low concentration groups （8，4，2 μmol/L）. Normal group and high glucose group were treated with low glucose DMEM medium （containing 5.5 mmol/L glucose ，similarly hereinafter ） and high glucose DMEM medium （containing 33.3 mmol/L glucose ， similarly hereinafter ）for 48 h，respectively. The cells in osmotic pressure control group were cultured in low glucose DMEM medium containing 27.5 mmol/L mannitol for 48 h. In DMDD groups ，cells were cultured in high glucose DMEM medium for 24 h， and then in high glucose DMEM medium containing corresponding concentration of DMDD for 24 h. At the end of cell culture ，MTT metho d was used to detect the cell survival rate. The activities of ROS ， GSH-Px and LDH in cellsupernatant were detected by using related kits. ELISA assay was used to detect the levels of TNF-α and IL-6 in cell supernatant. Cell apoptosis was d etected by acridine orange/ethidium bromide （AO/EB）staining. Western blot assay was used to detect the expression of apoptosis related proteins （cleaved caspase- 3，Bcl-2，Bax）and the phosphorylation level of nuclear factor κB （NF-κB）/NF-κB suppressor protein α（IκBα）signaling pathway related proteins （NF-κB p65，IκBα）. RESULTS ：Compared with the normal group ，survival rate ，the activity of GSH-Px and protein expression of Bcl- 2 in high glucose groups were decreased significantly（P＜0.01）；the activities of ROS and LDH ，the levels of TNF-α and IL-6，the protein expression of Bax and cleaved caspase-3，and the phosphorylation level of NF-κB p65 and IκBα were increased significantly（P＜0.01）；the cells showed orange yellow fluorescence ，and the number of cells with fuzzy morphology increased significantly ，showing an obvious apoptotic state. There was no statistical significance in above indexes of osmotic pressure control group compared with normal group. Compared with high glucose group ，the activities or levels of above indexes （except for cell survival rate an LDH activity in low concentration group ）were reversed significantly in DMDD groups （P＜0.05 or P＜0.01）；the orange yellow fluorescence in the cells decreased significantly ，and the cell morphology was relatively complete. CONCLUSIONS ：DMDD can significantly improve H9c2 myocardial cell injury induced by high glucose ；the mechanism of which may be associated with suppressing oxidative stress and inflammatory response ，regulating the expression of apoptosis related protein and NF-κB/IκBα pathway related protein.
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Objective To investigate the effect of basic fibroblast growth factor (bFGF) on autophagy of nerve cells in rats after traumatic brain injury (TBI).Methods A total of 120 healthy adult male SD rats were randomly divided into sham group,TBI + vehicle group,and TBI + bFGF group by random number table method,with 40 rats in each group.PinPointTM Precision Cortical Impactor was used to simulate the pathological damage after TBI.In the sham group,the dura was exposed without impact.In the TBI + bFGF group,250 μg/kg of human recombinant bFGF was given in the nasal cavity 1 hour before the modeling,while in the sham group and TBI + Vehicle group,the same amount of saline was given in the nasal cavity 1 hour before the modeling.The necrotic cells were observed by propidium iodide(PI) staining 6 hours after injury.The effect of bFGF on the nerve function after TBI in rats was evaluated with modified neurological severity score (mNSS) 24 hours after injury.The water content of brain tissue was measured by dry and wet method 48 hours after injury.The ratio of Beclin-1,P62 protein and microtubule-associated protein 1 light 3 (LC3)-Ⅱ/Ⅰ protein was detected by western blot.The volume of brain injury was calculated by integral method of brain tissue section.The positive neuron specific nuclear protein (NeuN) cells were observed by immunofluorescence staining.The apoptotic cells were observed by TUNEL.Results Compared with the sham group [(4.0 ± 1.2) ％],the percentage of necrotic cells in TBI + vehicle group [(54.3 ± 10.1) ％] and TBI + bFGF group [(34.5 ± 10.5) ％] increased significantly (P ＜ 0.05),but the percentage of necrotic cells in TBI + bFGF group increased less than that in TBI + vehicle group (P ＜ 0.05).Compared with the sham group [(0.3 ± 0.5)points],the mNSS in the TBI + vehicle group [(5.8 ±0.8)points] and TBI + bFGF group [(4.7 ± 1.1) points] were significantly increased (P ＜ 0.05),but the mNSS of TBI + bFGF group was lower than that of TBI + vehicle group (P ＜ 0.05).Compared with the sham group [(76.7 ± 0.7) ％],the water content of brain tissue of TBI + vehicle group [(79.2 ± 0.5) ％] and TBI + bFGF group [(78.4 ± 1.0) ％] were significantly increased (P ＜0.05),but the water content of TBI + bFGF group was significantly lower than that of TBI + vehicle group (P ＜0.05).Compared with the sham group,protein expression of Beclin-1 and LC3-I/Ⅰ in TBI + vehicle group and TBI +bFGF group were significantly improved (P ＜ 0.05),P62 protein expression was significantly decreased (P ＜ 0.05),the volume of brain tissue injury was significantly increased (P ＜ 0.05),the number of positive NeuN cells increased significantly (P ＜ 0.05),and the number of apoptotic cells and apoptotic cells were significantly increased (P ＜0.05).Compared with the TBI + Vehicle group,the up-regulation of Beclin-1 protein and LC3-Ⅱ/Ⅰ protein ratio was obviously inhibited in the TBI + bFGF group (P ＜ 0.05),the down-regulation of P62 protien was significantly suppressed,the volume of brain tissue injury was significantly decreased,and the number of positive NeuN cells and apoptotic cells was significantly reduced (P ＜ 0.05).Conclusion The bFGF can significantly inhibit excessive autophagy in rats after TBI,reduce brain edema,reduce cell apoptosis and necrosis,and improve neural function.
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Aim To investigate the effect of the extract of Averrhoacarambola L.root (EACR)on renal func-tion in diabetic mice and its anti-oxidative action. Methods Diabetic mice were established by tail vein injection with 120 mg·kg-1 streptozotocin (STZ)and were divided into 5 groups:model control group,val-sartan control group,and low-,middle-,high-dose of EACR groups (300,600,1 200 mg·kg-1 ).And 10 normal mice consisted of normal control group.The fasting blood glucose (FBG)of mice was detected be-fore and after administration of drugs.After last admin-istration,the blood and urine samples were collected for creatinine (Cr),urea nitrogen (BUN),urine and 24 h urinary protein determination.The activities of superoxide dismutase (SOD ),glutathione peroxidase (GSH-Px)and malonaldehyde (MDA)content were determined using kits.HE staining was conducted to observe the pathological changes of kidney tissues. ELISA method was utilized to detect the contents of catalase (CAT)and reactive oxygen species (ROS ). The expressions of Cyto-C,AIF and caspase-3 proteins in kidney tissues were analyzed by Western blot.Re-sults Compared with model group,the serum bio-chemical indexes and 24 h urinary protein of valsartan and moderate-,high-dose of EACR groups were de-creased with statistical significance (P<0.05 ).After the treatment, the MDA content was decreased by EACR treatment,and SOD,GSH-Px and CAT activities were enhanced.Meanwhile the expressions of ROS, Cyto-C,AIF and caspase-3 were down-regulated.The pathological changes of kidney tissues were ameliorated by EACR through HE results.Conclusions The ex-tract of Averrhoacarambola L.root can decrease the se-rum levels of Cr and BUN,reduce the MDA and ROS contents in kidney tissue and enhance the activities of SOD,GSH-Px and CAT,down-regulate the expres-sions of Cyto-C,AIF and caspase-3 proteins in kidney tissues,elevate the anti-oxidative effect of kidney. Therefore,the renal function of diabetic mice is melio-rated.
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BACKGROUND:It is confirmed that astrocytes can differentiate into neurons by Neurogenin2 gene regulation, suggesting that Schwann cells may also differentiate into neurons by gene regulation. OBJECTIVE:To evaluate the feasibility of Schwann cells differentiating into neurons by Neurogenin2 gene regulation. METHODS:Rats Schwann cells were isolated, purified and identified. Then the Schwann cells were transfected with Neurogenin2 via green fluorescent protein gene-plentivirus. To induce neuronal differentiation, the Schwann cells were cultured in serum-free Dulbecco’s modified Eagle’s medium containing epidermal growth factor, basic fibroblast growth factor and brain-derived neurotrophic factor for 2 weeks. The morphology of induced cells was observed by microscope, and myelin basic protein and neuron-specific enolase were detected by immunocytochemistry. RESULTS AND CONCLUSION:After transfection with Neurogenin2 via green fluorescent protein gene-plentivirus and induced differentiation, immunofluorescence assay demonstrated that 12.56%of the induced cellexpressed neuron-specific enolase, but the control group did not express neuron-specific enolase. Neurogenin2 gene-transfected Schwann cells can express neuron-specific enolase, suggesting Neurogenin2 gene may regulate transdifferentiation of Schwann cells into neurons.
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BACKGROUND: Cytoskeleton is a carrier of cell growth, and its pore caliber is one of the most important factors to affect the curative effect of tissue engineered spinal cord.OBJECTIVE: To explore the optimal pore size of poly lactic-co-glycotic acid (PLGA) scaffolds for tissue engineered spinal cord by in vitro culture of neural stem ceils (NSCs) and various pore sizes of PLGA scaffolds.METHODS: 50 μL (cell number 10~(10)/L)NSCs suspension at passage 1 was separately seeded on 200-300 pm, 400-500 μm PLGA stant for 7 days. Two sorts of tissue engineered spinal cord were constructed in vitro. Thirty rat models of spinal cord injury were established, and then assigned to 3 groups. The detect sites of these models were filled with above-mentioned spinal cord immediately, but the blank control was not treated with any material. The cells growth and proliferation implanted on PLGA were observed by phase contrast microscope and scanning electron microscope. Relative number of NSCs in two tissue engineered spinal cords was measured by MTT assay. The effects of transplantation with tissue engineered spinal cord were evaluated by the BBB scala.RESULTS AND CONCLUSION: Neural stem cells implanted on different pore size scaffolds were seen growing by phase contrast microscope and scanning electron microscope, with good histocompatibility. After 7-day coculture, absorbance was similar between 200-300 pm PLGA and 400-500 pm PLGA groups (P > 0.05). These indicated that the pore size had no effects on NSC number. At week 4 following transplantation, in the blank control group, neural function was recovered to different degrees in the 200-300 μm PLGA and 400-500 μm PLGA groups. BBB motor functional score was significantly increased (P < 0.05). The pore size of 200-300 μm utilized in fabriceting tissue engineered spinal cord has the best transplantation effect as compared to others.
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Objective This study was designed to determine the influence of acute ethanol intoxication (AEI) on brain edema and aquaporin-4(AQP-4) levels after traumatic brain injury(TBI) in rots. The underlying mechanism was also investigated. Method Severe traumatic brain injury models were made using the Feeny method; acute ethanol intoxication models were established by gavagy. One hundred and ninety-two male SD rats were randomly divided(random number) into four groups, namely the sham operation group(A ), the acute ethanol intoxication group( B ), the traumatic brain injury group(C) and the combination of acute ethanol intoxication with traumatic brain injury group(D). Each group was further divided into four sub-groups according to the time interval between injury and death of the rats. After brain tissue was fixed by affusing paraformaldehyde, the expression of AQP-4 was detected by immunohistochemistry. Water content was detected by dry-wet analysis, and AQP-4 mRNA and protein were detected by RT-PCR and western blotting respectively after the brain tissue was got by rapid decapitation. Data were analyzed by one-way ANOVA. Results The water content of brain tissue and expression level of AQP-4 were not significantly different between groups A and B( P > 0.05); however both were significantly increased in groups C and D relative to group A( P < 0.05). The water content of brain tissue in group D increased by mere than that in group C( P < 0.05), while the expression level of AQP-4 in group D was lower than that in group C(P<0.05). Conclusions Acute ethanol intoxication inhibited the expression of AQP-4,which induced a more severe cerebral edema after traumatic brain injury.
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Objective To investigate the effects of naloxone hydrechloride on neuronal cell apoptosis and cerebral edema induced by experimental brain injury.Method Animal model of brain injury was established by free-falling method of Fecney.Totally 100 SD rats were randomly divided into sham operated group(n=20),op-erated comparison group(n=40),and naloxone hydrochloride treatment group(n=40).The naloxone hy-drochloride treatment group were subdivided into four sub-groups,and naloxone hydrochloride was injected at 30 min,6 h,24 h and 48 h after brain injury respectively,while the comparison groups were injected with physiologi-cal saline at the same time point.The rats were sacrificed 7 deys after the injury.Neuronal cell apoptosis were de-tected by terminal-deoxynucleotidyl transferase mediated nick end labeling(TUNEL),and water content of brain tis-sue was examined with dry-wet methods.Data were analyzed by using spss 10.0 saftware package statistical analysis was carried out by using variance analysis.Results Gompared to the sham operated group,neuronal cell apopto-sis and water content of brain tissue were increased significantly in operated comparison group(P＜0.05).Com-pared to the operated comparison group,neuronal cell apoptosis and water content of brain tissue were decreased significantly in every naloxone hydirochloride treatment group(P＜0.05).Every sub-groups compared in naloxone hydrochloride treatment group,neuronal cell apoptosis of early injection group(30 min and 6 h after injury)were decreased significantly than late injection group(24 h and 48 h after injury,P＜0.05),but the water content of brain tissue had no difference between the early injection group and the late injection group(P＞0.05).Conclu-sions Naloxone can carry out its protective function to injuried neurecyte through alleviating neuronal cell apopto-sis and hydrecepahalus.and the effect was better as early as it used.
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Objective To investigate the value of subdural intracranial pressure (ICP) monito-ring in post-operative patients with severe brain injury. Methods A total of 100 patients with severe brain injury treated with craniotomy were randomly divided into ICP monitoring group (n=50) and rou-tine treatment group (n = 50). In ICP monitoring group, the treatment methods were adjusted according to the changes of ICP, whereas the patients in routine treatment group underwent general treatment ac-cording to standard neurosurgical protocol. Results Patients in ICP monitoring group received mannitol for eight days, with the average dosage of 950 g. Marmitol was administered to patients in routine treat-ment group for 12 days, with average dosage of 1 450 g. There was statistical difference in aspects of time duration and mannitol dosage between two groups (P <0.01). Of all patients in ICP monitoring group, four were found with electrolyte disturbance (8%), seven with acute renal failure (14%), four with stress ulcer (8%) and eight with pulmonary infection (16%). The corresponding numbers of patients in routine treatment group were nine (18%), 14 (28%), five (10%) and nine (18%), respectively. The occurrence of electrolyte disturbance and acute renal failure between two groups showed significant statistical difference (P < 0.05), while the occurrence of stress ulcer and pulmonary infection were be-yond of statistical difference between two groups (P > 0.05). The post-operative initial ICP level was positively correlated with mortality rate (P <0.01). All patients were followed up for three months post-operatively. In ICP monitoring group, 27 patients (26%) obtained good prognosis without any disability (54%), 13 were under mild disability, two (4%) under severe disability, three (6%) under vegeta-tive state and five (10%) died . In the routine treatment group, 17 patients (34%) were with good prognosis without any disability , six (12%) with mild disability , six (12%) with severe disability, eight (16%) under vegetative state and 13 (26%) died. The ICP monitoring group had better prognosis than the routine treatment group(P < 0.05). Conclusion Continuous ICP monitoring postoperatively in severe brain injury patients is valuable in reducing mortality, complication and improving the prognosis.
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Objective To dynamically observe the effect of mild hypothermia on concentration of plasma S-100B protein in patients with acute severe brain injuries so as to further explore its role in treat-ment of acute severe brain injury. Methods A total of 120 patients with acute severe brain injuries were randomly divided into mild hypothermia group and general group. The patients in mild hypothermia group were treated with mild hypothermia besides conventional therapy, with maintenance of rectal tem-perature at 33℃-35℃ for 3-5 days. Serial concentration of S-IOOB protein in serum was measured in all patients from 6 hours to 6 days after hospitalization. GOS evaluation was done three months after treat-ment. Results The concentration of S-100B protein in serum of mild hypothermia group and general group was significantly higher than of normal group (P <0.05), with significant lower level in mild hypo-thermia group than general group(P <0.05). Mild hypothermia could improve prognosis of patients with acute severe brain injury. Conclusions Early use of mild hypothermia can decrease concentration of S-100B protein in serum, protect neurofunction and improve prognosis, as may be related to its function in alleviating damnification brain cell inflammation reaction mediated by S-100B protein.