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OBJECTIVE: To compare the therapeutic effect of electroacupuncture (EA) of "Shenmen" (HT 7, Yuan point of the Heart Meridian), "Zhizheng" (SI 7, Luo point of the Small Intestine Meridian) and "Xinshu" (BL15, Back-shu point) on the expression of nerve growth factor (NGF) and its receptor, tyrosine kinase A (TrkA) in the cerebral cortex of myocardial ischemia (MI) rats. METHODS: A total of 70 male SD rats were randomized into sham operation (sham, n=10), model, HT 7, SI 7, and BL15 groups (n=15 in each of the latter 4 groups). The MI model was established by ligation of the anterior descending branch of the left coronary artery. EA (1 mA, 2 Hz) was respectively applied to HT 7, SI 7 and BL15 for 15 min, once per day for 1 week. Immunohistochemistry and real-time fluorescent quantitative PCR were used to detect the expression of NGF and TrkA proteins and genes in the cerebral cortex. RESULTS: Compared with the sham group, no significant changes were found in the number of NGF immune-reaction (IR)-positive and TrkA IR-positive cells, and the expression levels of NGF mRNA and TrkA mRNA in the model group (P>0.05). After EA intervention, the number of NGF and TrkA IR-positive cells and the expression of NGF mRNA and TrkA mRNA in each of the 3 EA groups were significantly increased relevant to the model group (P<0.01, P<0.05). The effect of EA at BL 15 was significantly superior to that of EA at HT 7 and SI 7 in increasing the number of NGF and TrkA positive cells and up-regulating the expression of their mRNAs (P<0.01, P<0.05). CONCLUSION: EA at HT 7, SI 7 and BL 15 can increase the levels of expression of NGF and TrkA proteins and mRNAs in the cerebral cortex of subacute MI rats and the effects of EA-BL 15 are obviously superior to those of EA-HT 7 and EA-SI 7, suggesting a relative specificity of the effect of EA at different acupoints.
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Objective@#To study the effects of nerve growth factor (NGF) on the proliferation, osteogenic differentiation and mineralization of type 2 diabetic mice bone marrow stromal cell (BMSC), providing basis for clinical application of NGF.@*Methods@#Three 8-week-old male db/db mice and two 8-week-old male C57BL/6J mice were used in the study. BMSC derived from femur were cultured though adherence method. BMSC of C57BL/6J mice and db/db mice was divided into normal group and diabetic group to conduct the osteogenic potential experiment, named experiment one. In experiment two, diabetic BMSC was divided into 3 groups: diabetic control group, NGF group, and K252a+NGF group [K252a was the inhibitor of tyrosine kinase A (TrkA), which was the high affinity receptor of NGF], to investigate effect of NGF on osteogenic potential of diabetic mice BMSC. After seeding BMSC, K252a was added into K252a+NGF group, then NGF was added 30 min later. NGF was added into NGF group and K252a+NGF group, but not diabetic control group. The proliferation of BMSC at 1, 3, 5 and 7 d in experiment one and the proliferation of BMSC at 1, 2 and 3 d in experiment two were evaluated through methyl thiazolyl tetrazolium, and the level of alkaline phosphatase (ALP) at 3, 5 and 7 d in both experiments were measured. After being osteogenic induced for 14 d, mineralized nodules in both experiments were quantitated by alizarin red calcium stain. Five holes were set in every group, and all experiments were repeated 3 times.@*Results@#The BMSC proliferation of diabetic group was significantly higher than that of the normal group at 3, 5 and 7 d (P<0.05). After being osteogenic inducted for 3, 5 and 7 d, ALP level of diabetic group were significantly lower than that of normal group (P<0.05). After being osteogenic inducted for 14 d, calcium nodule count of diabetic group [(23.1±6.4) nodule/field] were significantly lower than that of normal group [(36.9±7.9) nodule/field](P<0.05). At 1, 2 and 3 d, BMSC proliferations of diabetic control group, NGF group and K252a+NGF group were not statistically different (P>0.05). After being osteogenic inducted for 3 and 5 d, ALP level of NGF group was significantly higher than that of diabetic control group (P<0.05). After being osteogenic inducted for 3, 5, and 7 d, ALP level of K252a+NGF group was significantly lower than that of NGF group (P<0.05) and diabetic control group (P<0.05). After being osteogenic induced for 14 d, calcium nodule count of NGF group [(45.2±6.8) nodule/field] was significantly more than that of diabetic control group [(23.1±6.4) nodule/field](P<0.05); while calcium nodule count of K252a+NGF group [(18.0±4.5) nodule/field] was significantly less than that of NGF group (P<0.05) and diabetic control group (P<0.05).@*Conclusions@#The differentiation and mineralization of type 2 diabetic mice BMSC was significantly reduced. NGF promoted the osteoblastic differentiation and mineralization of diabetic mice BMSC in viro though combining with TrkA.
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BACKGROUND: Nerve growth factor (NGF) promotes the survival and differentiation of vertebrate neurons, and their actions are mediated by two classes of cell surface receptors: tyrosine kinase A receptor (TrkA) and p75 neurotrophic receptor (p75NTR). We evaluated the role of NGF receptors in neuronal survival and the physical interactions between them. METHODS: Organotypic hippocampal slices were obtained from 5 to 7-day-old rat pups and were grown for 14 days in vitro. The expression of the TrkA and p75NTR was evaluated by the western blot and immunohistochemical methods. The neuroprotective effect of NGF on the blocking of antibody-induced neuronal cell death was tested by the application of NGF (0, 50 and 150 ng/ml) to the culture media in the presence of 200 ng/ml of blocking antibodies against TrkA and p75NTR. Functional interactions between the two receptors were examined using the immunoprecipitation method. RESULTS: TrkA and p75NTR were co-expressed in the principal neurons of the hippocampal slice culture, and the expression level was increased time dependently until 14 days of culture. The blocking antibody against each receptor induced neuronal damage in time and dose-dependent manners. NFG delayed or prevented the blocking antibody from inducing neuronal damage. Results from the immunoprecipitation experiment showed physical interactions between the two NGF receptors. CONCLUSIONS: Our results indicate that the co-expressed NGF receptors, TrkA and p75NTR, might have protective roles in the survival of neuronal cells through the cooperative interactions between them.