Effects Of Iridiod Glycosides Of Paederia Scandens Drug-containing Serum On Dorsal Root Ganglia Neuron In Neonatal Rats In Vitro

Pain is an unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage. Iridiod glycosides of Paederia scandens (LOUR.) MERRILL (Rubiaceae) (IGPS) is a major active component isolated from traditional Chinese herb Paederia scandens (LOUR.) MERRILL (Rubiaceae) which are contain paederoside, asperuloside, scandoside et al. Paederia scandens was described to use as analgesic, anti-inflammatory, and to treat bruise and rheumatism through eliminating heat and excreting dampness in terms of traditional Chinese medicine. The initial research demonstrated that IGPS had antinociceptive effects on different animal pain models. This study is intended to investigate the effects of Iridiod glycosides of paederia scandens drug-containing Serum on dorsal root ganglia neuron in neonatal rats in vitro. Effective downstream target of IGPS drug-containing serum could be found out. The main results are as follows:1 Neonatal rat dorsal root ganglion neurons purified and culturedDRGs were dissected from the neonatal (1-to 4-d old) Wister rats in axenic conditions. The ganglia were enzymatically digested half an hour at 37℃by collagenase typeâ…¡(0.3mg/ml: Sigma, USA) and dispase (0.9mg/ml: Invitrogen, USA).Neurons were cultured in 45% MEM and 45% Ham's F12 (Invitrogen, USA) supplemented with 10% heat-inactivated fetal calf serum. 5-fluoro-2'-deoxyuridine (5-FUDR, 10 mM) was added into feeding medium to purify DRGs. DRGs can survive up to two weeks in good condition. The expression of neuron-specific enolase (NSE) was detected by immunohistochemistry to identification of the DRG cells.2 DRG neurons injury modelDRG neurons were treated with sodium nitroprusside (SNP) at the fifth day with different concentration (2000, 200, 20μmol/L).Cells were measured by trypan blue viability test. The increase of DRG neurons death rate (50%) is proportional to the rise of the concentration of the SNP.3 Preparation IGPS drug-containing serumThirty male rats were randomly divided into three equal groups according to their body weight. Rats in the three groups were orally administered low (70 mg/kg), intermediate (140 mg/kg) and high (280 mg/kg) doses of IGPS one time each day (The doses were confirmed by previous studies in vitro). Rat blood was obtained from the carotid arteries 1 h after the final medicine administration for preparation of drug-containing serum. All samples went through separation and deactivation at 56°C for 30 min, suction filtration with a 0.22μm filter for sterilization, dilute 10.0 ml of serum to 100.0 ml with the feeding medium, freezing at-20°C for future use.4 IGPS drug-containing serum ameliorates SNP-induced dorsal root ganglia neuron injury in neonatal rat in vitroWe studied the effects of IGPS drug-containing serum on DRG neuron in neonatal rats in vitro. Cells were measured by trypan blue viability test and MTT assay. Intracellular Ca²⁺ level were quantified by single cell fluo3 fluorescence imaging. Inducible Nitric oxide synthase (iNOS) activity and Nitric oxide metabolites were measured by UV spectrophotometer. 3'-5'-cyclic guanosine monophosphate (cGMP) protein expression were investigated by radioimmunoassay and mRNA expression of iNOS, protein kinase G type I (PKGI, including PKGΙαand PKGIβ) were analyzed by reverse transcription-polymerase chain reaction (RT-PCR).The results showed that IGPS drug-containing serum (140,280mg/kg, ig) significantly alleviated SNP-induced mechanical mortality rate, which accompanied with markedly decreased spinal NOS activity, NO production and cGMP content. At the same time, IGPS-containing serum inhibited [Ca²⁺]i increases in DRG neurons(280mg/kg,ig), inhibited mRNA expression of iNOS and PKGΙαand PKGIβof the spinal cord tissue(140,280mg/kg,ig). These results suggested that IGPS drug-containing serum possesses protective effects on SNP injured DRG neurons by inactivating Ca²⁺-NO/cGMP/PKG signal transduction pathway which plays a crucial role.Conclusions:In summary, our results demonstrated that IGPS drug-containing serum had protective effects on injured DRG neurons. The mechanisms of its abirritation may be related to its inhibition of Ca²⁺-NO/cGMP/PKG signal pathway.