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Objective:To observe any effect of electroacupuncture (EA) on the expression of phosphorylated extracellular signal-regulated protein kinase (p-ERK1/2) and phosphorylated cyclic adenosine monophosphate response element binding protein (p-CREB) in the spinal dorsal horns of diabetics experiencing neuropathic pain.Methods:Eight rats were randomly selected from 30 healthy male Sprague-Dawley rats as the normal group (N), and the remaining twenty-two rats were treated with a single high-dose intraperitoneal injection of streptozotocin (STZ) to establish a neuropathic pain model. The rats modeled successfully were randomly divided into a model group (M, n=8) and an EA group ( n=8). In the EA group, electroacupuncture was applied at the bilateral Hou san li and Kunlun acupoints starting on the 15th day after the STZ injection. The daily sessions lasted 30 minutes for 1 week. Body weight (BW), fasting blood glucose (FBG) and paw withdrawal latency (PWL) were observed before the STZ injection and on the 7th, 14th, and 21st days afterward. The expression of p-ERK1/2 and p-CREB in the dorsal horns of the rats′ spinal cords was detected using western blotting. The count of p-CREB-positive cells in the dorsal horns and their co-localization with neurons was detected using immunofluorescence. Results:In comparison with the N group, the average BW of the M group on the 7th, 14th and 21st days after the STZ injection was significantly lower, while the average FBG was significantly higher. There was no significant difference between the M and N groups in the average PWL on the 7th day after the STZ injection, but it had decreased significantly in the M group on the 14th and 21st days. Compared with the M group, the average PWL of the EA group was significantly longer on the 21st day after the injection. The expression of p-ERK1/2 and p-CREB protein in the spines of the M group was significantly higher than in the N group. p-CREB positive cells were more numerous in the M group compared with the N group, while in the EA group they were fewer. P-CREB was co-located with neurons in the spinal dorsal horn.Conclusion:EA can alleviate neuropathic pain effectively, perhaps by inhibiting the expression of p-ERK1/2 and p-CREB in the dorsal horns of the spinal cord.
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Objective:To observe the effects of Albiziae Flos （AF） and Polygalae Radix （PR） alone and their combination on the improvement of depression-like behavior in rats with chronic unpredictable stress （CUS） as well as on hippocampal ultrastructure and the expression of cyclic adenosine monophosphate response element binding protein （CREB） and nicotinamide adenine dinucleotide phosphate oxidase 2 （NOX2）， to explore their action mechanisms. Method:Seventy-two Wistar rats were randomly divided into the normal group， model group， AF group， PR group， AF-PR group， and fluoxetine group. Rats in all groups except for the normal group were exposed to CUS and separated feeding to induce depression. Since the first day of modeling， rats in the AF group， PR group， AF-PR group were provided with the corresponding decoction containing 1.05 g·kg<sup>-1</sup> total crude drug by gavage， the ones in the fluoxetine group with 2.1 mg·kg<sup>-1</sup> fluoxetine hydrochloride aqueous solution， and those in the normal group and model group with the distilled water， for 28 successive days. The open field test and forced swimming test were performed 1 d before modeling and 7， 14， 21， 28 d after modeling， respectively. The morphological changes in hippocampus were observed under an electron microscope on the 28<sup>th</sup> day. The superoxide dismutase （SOD） and malondialdehyde （MDA） levels in hippocampus were detected by ultraviolet spectrophotometry， and the expression levels of CREB and NOX2 were detected by real-time polymerase chain reaction （Real-time PCR） and Western blot. Result:The behavioral experiment results showed that the number of horizontal activities and sugar water consumption in the model group declined as compared with those in the normal group， while the immobility time in the forced swimming test was prolonged （<italic>P</italic><0.05， <italic>P</italic><0.01）. Compared with the model group， the AF group， PR group， and AF-PR group exhibited elevated number of horizontal activities， increased sugar water consumption but shortened immobility time in the forced swimming test （<italic>P</italic><0.05， <italic>P</italic><0.01）. Compared with the AF group or PR group， the AF-PR group showed significantly different behavioral indexes （<italic>P</italic><0.05）. Morphological results showed that the mitochondria of the model group were obviously swollen and the ultrastructure of the hippocampus was destroyed. By contrast， the hippocampal ultrastructure in each administration group was close to normal. The comparison with the normal group revealed that the activity of SOD in the hippocampus of the model group was significantly reduced， whereas the content of MDA was elevated （<italic>P</italic><0.01）. Compared with the model group， the AF group， PR group， and AF-PR group displayed increased activity of SOD and decreased content of MDA in the hippocampal tissues （<italic>P</italic><0.05， <italic>P</italic><0.01）. Compared with AF or PR alone， the herbal pair AF-PR resulted in significant differences in the above-mentioned indexes （<italic>P</italic><0.05， <italic>P</italic><0.01）. The results of Real-time PCR and Western blot demonstrated that NOX2 expression in the hippocampus of the model group was up-regulated in comparison with that in the normal group， while the CREB expression was down-regulated （<italic>P</italic><0.05， <italic>P</italic><0.01）. Compared with the model group， the AF group， PR group， and AF-PR group all showed diminished NOX2 expression but elevated CREB expression in the hippocampal tissues （<italic>P</italic><0.05， <italic>P</italic><0.01）. The protein expression levels of NOX2 and CREB in the AF group or PR group were significantly different from those in the AF-PR group （<italic>P</italic><0.01）. Conclusion:AF and PR alone and their combination improve the depression-like behavior of rats exposed to CUS， which may be related to the reduction of oxidative stress， the up-regulation of CREB expression， and the down-regulation of NOX2 expression in hippocampus.
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Objective:To optimize proportion of couplet medicine of Bupleuri Radix and Scutellariae Radix in the treatment of anti-depression, and to explore the possible antidepressant mechanism of this couplet medicine. Method:The dosages of Bupleuri Radix and Scutellariae Radix in the 2015 edition of Chinese Pharmacopoeia were taken.According to U7(72) uniform design table, Bupleuri Radix and Scutellariae Radix were carried out into 7 groups.The chronic unpredictable mild depression model mice were induced by intragastric administration of decoction of this couplet medicine and the antidepressant effect was observed by the behavior tests, which included sucrose preference test, tail suspension test, forced swimming test and open field test(crossing scores).The regression equations were established by selecting the effective indexes.The experiments of retest were taken to check the results and the possible antidepressant mechanism was primarily investigated by measuring the phosphorylation level of cyclic adenosine monophosphate(cAMP)-response element binding protein(CREB) and expression of brain-derived neurotrophic factor(BDNF). Result:Compared with the blank group, sucrose preference rate of the model group was significantly decreased(PPPPPPPPPPPPPPConclusion:Compatibility of Bupleuri Radix and Scutellariae Radix can ameliorate depressive-like behavior of model mice, and the best antidepressant compatibility proportion of Bupleuri Radix and Scutellariae Radix is 1:1, the optimal amounts of them are about 5 g.The antidepressant effect may relate to promoting phosphorylation level of CREB and the expression of BDNF in the hippocampus.
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Objective: To investigate the neuroprotective effect and mechanism of tetrahydroxy stilbene glucoside (TSG) on β-amyloid protein 25-35 (Aβ25-35)-induced neuron synapses damage. Method: Primary neurons were isolated and purified from cerebral cortex of suckling mouse. Then neurons were divided into control group, model group (incubation with Aβ25-35) and TSG groups (after incubation with Aβ25-35, add 6.25, 12.5, 25, 50, 100 μmol·L-1 TSG). Cell counting kit-8 (CCK-8) and Lactate dehydrogenase (LDH) methods were used to observe the viability of neuron, immunocytochemical staining was performed to determine the expressions of synapsin-1 (SYN-1), and the concentration of postsynaptic density-95 (PSD-95) and synaptophysin (SYP) were detected by enzyme-linked immunosorbent assay (ELISA) method. The level of cyclic adenosine monophosphate response element binding protein (CREB) and brain-derived neurotrophic factor (BDNF) mRNA were determined by reverse transcription-polymerase chain reaction (RT-PCR) and the level of CREB, Phosphorylated CREB (p-CREB) and BDNF proteins were determined by immunocytochemical staining or Western blot (WB). Result: Compared with normal group, the cell survival rate of model group was significantly reduced, LDH release was significantly increased (PPPPPPP-1,25 μmol·L-1 TSG can significantly enhance the expression of SYN-1(PPPPConclusion: TSG possesses the neuroprotective effect on Aβ25-35-induced neuron synapses, the mechanism may be associated with the activation of CREB/BDNF signaling pathway.
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Objective To explore the effect of the exogenous phosphorylation cyclic adenosine monophosphate response element bind-ing protein (pCREB) antibody on the expression of apoptotic regulated genes (Bcl-2 and c-Jun) in hippocampus after status convulsivus (SC), to elucidate the role and regulation mechanism of pCREB in convulsive brain injury. Methods Seizures were induced in 24 adult Wi-star rats with lithium-pilocarpine intraperitoneal injection (SC group), another 24 rats were as the normal controls (NC group). Each group was divided into no injection subgroup, normal saline injection subgroup and anti pCREB subgroup according to the injection contents of lat-eral ventricle, with 8 cases in each group. They were sacrificed 6 hours after injection. Both the protein and mRNA expression of Bcl-2 and c-Jun in bilateral hippocampus were detected by immunohistochemistry and in situ hybridization, respectively. Results There was no signifi-cant difference in Bcl-2 protein/mRNA expression among 3 subgroups in the NC group (P>0.05). In the SC group, the expression of Bcl-2 protein/mRNA were lower in the anti pCREB subgroup than in the no injection subgroup and normal saline injection subgroup (P<0.05). There was significant difference in c-Jun protein/mRNA expression among 3 subgroups in both NC group and SC group (P<0.001). The ex-pression of c-Jun protein/mRNA was higher in the normal saline injection subgroup and the anti pCREB subgroup than in the no injection group (P<0.05), especially in the anti pCREB subgroup (P<0.05). Conclusion Exogenous anti-pCREB antibody can down-regulate the ex-pression of Bcl-2 and up-regulate the expression of c-Jun in hippocampal cells after SC.
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Objective To investigate the effects of different duration of fluoxetine and escitalopram administration on depressive-like behavior and hippocampal BDNF expression in adult rats.Methods Ninety-six SD rats were randomly divided into twelve groups: (1)M 1 group: normal control for one week, (2)M2 group: CUMS +saline for one week, (3)M3 group: CUMS+fluoxetine for one week, (4) M4 group: CUMS+escitalopram for one week, (5) M5 group : normal control for two weeks, (6) M6 group : CUMS+ saline for two weeks, (7) M7 group : CUMS+fluoxetine for two weeks, (8)M8 group: CUMS+escitalopram for two weeks, (9)M9 group: normal control for three weeks,(10) M10 group: CUMS+saline for three weeks, (11)M11 group: CUMS+fluoxetine for three weeks, (12) M8 group: CUMS+escitalopram for three weeks.After CUMS procedures,rats in M2 group,M6 group and M10 group were injected with saline, M3 group, M7 group and M11 group were injected with fluoxetine, and rats in M4 group,M8 group,M12 group were injected with escitalopram.After one week of intervention,the openfield test and 1％ sucrose preference test were performed to evaluate depression-like behaviors in rats of M1 group, M2 group,M3 group and M4 group.After behavior test,rats were sacrificed and the hippocampi were isolated.The expression of BDNFmRNA was detected by using real-time quantitative PCR.After two weeks of intervention, rats in M5 group,M6 group, M7 group and M8 group underwent the same behavioral.After three weeks of intervention, rats in M9 group, M10 group, M11 group and M12 group underwent the same behavioral test.Results In the open-field test,total distance travelled in 10 minutes was significant difference among the following groups: M1 group ((3925.70±322.32) cm) vs M3 group ((1841.85±786.33) cm) ,M6 group ((1820.31±296.00) cm) vs M8 group ((4002.72± 1447.19) cm), M10 ((1961.66±919.16) cm) group vs M11 group ((3741.72± 1064.46)cm) ,M10 group ((1961.66±919.16) cm) vs M12 group ((4280.43±1187.05) cm).In the 1％ sucrose preference test,the difference of sucrose preference consumption was statistically significant (P＜0.05) among the following groups: M2 group ((56.23±7.49)％) vs M4 group ((70.55±4.96)％), M6 group ((60.22±8.81)％) vs M8 group ((75.08±4.15)％) ,M10 group ((60.26±7.20)％) vs M11 group ((73.88±7.73)％) ,M10 group ((60.26 ± ±7.20)％) vs M12 group ((73.52±7.58)％).The expression level of BDNF was significant difference among these groups: M2 group (0.66±0.14) vs M4 group (1.15±0.20) ,M10 group (0.90±0.15) vs M11 group (1.22± 0.09) ,M10 group (0.90±0.15) vs M12 group (1.48±0.20).Conclusion Both of fluoxetine and escitalopram can improve depression-like behaviors in rats and significantly increase the expression of the hippocampal BDNFmRNA.Compared with fluoxetine,escitalopram has a shorter onset time in the treatment of depression.It may be related with a rapid increase of the expression of BDNF mRNA.
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Objective To study changes of phosphorylated cyclic adenosine monophosphate(c-AMP) response element binding protein in hippocampus(CA1) of neonatal rats after cerebral hypoxic-ischemia(HI)and effects of gangliosides (GM1)on the p-CREB.Methods An animal model of neonatal hypoxic-ischemia brain damage was established. Changes of p-CREB in hippocampal CA1 was detected with immunohistochemical methods.Results The p-CREB levels in the CA1 of HI and GM1 groups increased transiently and then decreased quickly, but there was no significant difference between HI and GM1 group.Conclusion The p-CREB levels in the CA1 of HI group increase transiently and then decrease quickly after HI ;GM1 has little effect on p-CREB in the CA1 after HI.