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<p><b>OBJECTIVE</b>To examine the effects of the combination of quercetin (Q), cinnamaldehyde (C) and hirudin (H), a Chinese medicine formula on high glucose (HG)-induced apoptosis of cultured dorsal root ganglion (DRG) neurons.</p><p><b>METHODS</b>DRG neurons exposed to HG (45 mmol/L) for 24 h were employed as an in vitro model of diabetic neuropathy. Cell viability, reactive oxygen species (ROS) level and apoptosis were determined. The expression of nuclear factor of Kappa B (NF-κB), inhibitory kappa Bα(IκBα), phosphorylated IκBα and Nf-E2 related factor 2 (Nrf2) were examined using reverse transcription-polymerase chain reaction (RT-PCR) and Western blot assay. The expression of hemeoxygenase-1 (HO-1), interleukin-6 (IL-6), tumor necrosis factor (TNF-α) and caspase-3 were also examined by RT-PCR and Western blot assay.</p><p><b>RESULTS</b>HG treatment markedly increased DRG neuron apoptosis via increasing intracellular ROS level and activating the NF-κB signaling pathway (P<0.05). Co-treatment with Q, C, H and their combination decreased HG-induced caspase-3 activation and apoptosis (P<0.05 or P<0.01). The expressions of NF-κB, IL-6 and TNF-α were down-regulated, and Nrf2/HO-1 expression was up-regulated (P<0.05 or P<0.01). QCH has better effect in scavenging ROS, activating Nrf-2/HO-1, and down-regulating the NF-κB pathway than other treatment group.</p><p><b>CONCLUSIONS</b>DRG neurons' apoptosis was increased in diabetic conditions, which was reduced by QCH formula treatment. The possible reason could be activating Nrf-2/HO-1 pathway, scavenging ROS, and inhibition of NF-κB activation. The effect of QCH combination was better than each monomer or the combination of the two monomers.</p>
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Aim To further study the molecular mecha-nism of the herbs with hot nature on the regulational action on TRPV1 channel based on the 7900 Real-time PCR instrument. Methods 7900 PCR instrument was applied to detect the intracellular flurescence of TRPV1 channel in the dorsal root ganglions ( DRG ) neurons and the effect on the TRPV1 ’ s thermo-sensational functions of the selected 11 ingredients from hot herbs was explored. Results TRPV1 channels could be ac-tivated by gradually elevated temperature; the activa-tion process could be blocked by the TRPV1 specific blocking agent capsaizepine. Most of the ingredients from hot-nature herbs had the potential to up-regualate TRPV1 channel function. Conclusions The estab-lished TRPV1 channel detection system based on PCR instrument is suitable for the analysis of regulational functions of drugs on the heat-activated TRPV1 chan-nel;the functions of hot herbs may be related to the up-regualtional effects of its active ingredients on the TRPV1 channel which will further up-regulate energy metabolism.
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Objective To determine whether or not capsazepine(CPZ),a transient receptor po-tential vanilloid-1(TRPV1)antagonist,attenuates lidocaine-induced cytotoxicity on rat dorsal root ganglion (DRG)neurons in vitro.Methods Adequate DRG neurons from 3-day neonatal Wistar rats were obtained,cultured and purified in vitro.To achieve higher cell viability,we reduced the concen-tration of trypsin to 0.125% compared with others.The purified DRG neurons were incubated with 0,2.5,5,10,20 and 40 mmol/L lidocaine for 10 min,respectively,their viabilities were examined using Cell Counting Kit(CCK-8)assay,and the lethal concentration 50(LC50 )of lidocaine on DRG neurons was calculated.Then,the variation of lidocaine-induced cell viability at LC50 ,when 0,1,10 and 100 μmol/L CPZ were respectively added to the incubations,was examined with CCK-8 assay. Results The purified percentage of DRG neurons was as high as 91% after digesting by 0.125%trypsin and purifying in vitro.Cell viability of DRG neurons in group L1,L2,L3,L4,L5 was signifi-cantly down regulated compared with the control group,to be specific,that of L3,L4,L5 being re-markably lower than that of L1,that of L4,L5 lower than that of L2 and that of L5 lower than that L3 and L4.After lidocaine induced DRG neurons for 10 min,LC50 was 30 mmol/L;10 μmol/L and 100 μmol/L CPZ significantly reduced LC50 DRG neuron toxicity induced by lidocaine (P <0.05). The effect of 10 μmol/L CPZ had reached the maximal effect,decreasing the cell viability decrease from 50% to 35%.Conclusion The novel method in this experiment is effective to obtain good DRG neurons,the LC50 of lidocaine on rat DRG neurons is 30 mmol/L,and CPZ attenuates the cytotoxicity induced by lidocaine on rat DRG neurons.
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Objective To investigate the biophysical properties of mechanosensitive(MS) channels in cultured dorsal root ganglion neurons of neonatal rats. Methods MS channels current of cultured dorsal root ganglion neurons of neonatal rats were recorded using cell-attached and inside-out patch-clamp technique.The biophysical properties such as pressure response relationship,current voltage relationship,channel kinetics and ion selectivity were analyzed.Membrane stretch was achieved by the application of negative pressure(suction) to a patch-clamp electrode. Results One type of MS non-selective cation ion channels in the membrane patches tested in cultured dorsal root ganglion neurons of neonatal rats were recorded. Those channels were activated rapidly when suction was applied, and kept active during sustained application of negative pressure and quickly turned off when the suction was released.The MS channels exhibited a nearly linear current voltage relationship in the balance solution.The outward chord conductance was (96.2?3.6)pS (mV is between +40 mV and +60 mV) and the inward slope conductance was (62.5?0.4)pS (mV is between -60 mV and 0 mV). This kind of channels appeared to be outward rectifier.The average reversal potential was (-2.3?0.8)mV.The channel kinetics analysis indicated that suction could significantly increase the duration of short-openings and long-openings and decrease that of long-closings,with no effects on short-closings. Conclusion The results of this study could serve as a reference to the understanding of electric activity of DRG neurons.
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Objective In order to explore the mechanisms that regulate the expression of genes coding for cytoskeletal proteins during nerve regeneration. Methods In the present study, in situ hybridization was used to examine the changes of light(NF L),medium(NF M)and heavy (NF H)neurofilament protein subunits mRNA in L 4-6 dosal root ganglion (DRG) sensory neurons during nerve regeneration following a unilateral crush of the sciatic nerve. Results The hybridization signals of each neurofilament subunit mRNA were dramatic decrees in DRG sensory neurons post axotomy by light microscope.The signals of NF\|L and NF\|M mRNA were located in cytoplasm of neurons,whereas NF H mRNA was found in both nucleus and cytoplasm of neurons. Conclusion There are the different mechanisms of regulation of neurofilament subunit genes expression,the reduced neurofilament gene expression may represent a general response to axonal injury and plays an important role in effective nerve regeneration.
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The technique of double labelling with fluorescent tracers(FB-NY,PI-Bb)has.been used in the following three groups of experiments:1.in 5 rats,one fluorescenttracer was injected into the tibial nerve and the other was injected into the peronealnerve;2.in 14 rats,one fluorescent tracer was injected into the sural cutaneousnerve and the other was injected into the branches to the soleus and gastrocnemiusmuscles;3.in 13 rats,one tracer was injected into the tibial nerve and the otherinto the bladder wall.The sections of the L_(3-6),S_(1-3) dorsal root ganglion were stu-died with a olympus fluorescence microscope using UG-1 excitation filter system.In the first and second groups,double labelled neurons were observed in L_(4-6) dorsalroot ganglion.But in the third group,the double labelled neurons were found onlyin the L_6 segment.The type of the double labelled neurons was mainly small-sized(33.1%)and small-medium sized(40.9%).The findings indicated that,the primary afferent fibers branched towards theperiphery and supplied,with different branches,either two somatic areas or skinand deep structures,or,especially,visceral and somatic sensory fields.This resultsuggests that the peripheral dichotomization of these dorsal root ganglion cells mightconverge sensory inputs from the somatic(tibial nerve)and the visceral(bladderwall)fields and thus provides one of the structural basis for the referred pain andthe neuronal mechanism of acupunctural therapy by which stimulation of somaticstructures could regulate the activities of visceral organs.
ABSTRACT
Skin-visceral divergent projections of cholecystokinin(CCK)-containing dorsal root ganglion neurons were studied by combined technique of fluorescent double-labelling and immunohistochemistry. Fast Blue(FB) and Nuclear Yellow (NY) were injected into the coeliac ganglion and the cutaneous branches of left 9th-11th intercostal nerves, respectively. Three kinds of neurons labelled with fluorescein were observed in T_(9-11) dorsal root ganglia under Nikon fluorescence microscope with 365 nm excitation light: FB-labelled neurons with blue-fluorescing cytoplasm; NY-labelled neurons with yellow-fluorescing nucleus and double-labelled neurons with blue cytoplasm and yellow nucleus. The double-labelled neurons were found to be 2.8％ of total labelled neurons.The sections containing fluorescein labelled neurons were then stained by CCKimmunohistochemical procedure. Four kinds of neurons could be identified: NY-neurons, with CCK-immunoreactivity (NY+CCK); FB-neurons with CCK-immunoreactivity(FB + CCK);NY + FB neurons with CCK-immunoreactivity(NY + FB + CCK); Single CCK-positive neurons. NY + FB + CCK triple-labelled neurons accounted for approximately 11.5％ of NY + FB double-labelled neurons,and 0.4％ of all CCK-positive neurons.The findings clearly indicated that the peripheral processes of some sensory dorsal root ganglion neurons project divergently to both skin and visceral structure, and contain CCK. The present results suggest that the peripheral dichotomization of the dorsal root ganglion nearons might converge sensory inputs from both skin and visceral fields, and thus not only provide one of the structural basis for the referred pain but also reveal that CCK might play a mediation role in the skin-visceral reflection and referred pain.