Objective Dynorphins have advantages in powerful analgesic effect, high safety, no respiratory depression and no addiction, which is the emphasis of analgesic research at present. The aim of the article was to explore the expression of lentivirus-mediated rat prodynorphin gene in bone marrow mesenchymal stem cells( BM-MSCs) and contribute to the subsequent studies on bio-logical analgesia in cancer pain of rat model. Methods BM-MSCs were isolated and proliferated using the adherence screening meth-od, and further identified by flow cytometry, adipogenic and osteogenic differentiation experiments. The PDYN lentiviral vectors in rats were transfected into BM-MSCs after construction. The expression of green fluorescent protein (GFP) was detected under inversion fluo-rescence microscope and the best multiplicity of infection ( MOI ) of virus was screened by western blot. There are three groups in the ex-periment: blank group, experimental group ( PCDH-CMV-PDYN-EF1-copGFP ) and empty vector group ( PCDH-CMV-MCS-EF1-copGFP). PYDN gene was determined by qPCR and western blot, while DYN protein was detected by immunochemical method.Results BM-SMCs were in longspindle-shape and fibrocyte-like adherent growth, most in expression of CD29, CD44 and CD90, and a few in CD45. The oil red-O staining of the induced cells by adipogenic differentiation was positive. The mineralized nodules formed in the induced cells by osteogenic differentiation were orange after alizarin red staining. Flow cytometry detection showed the positive rates of CD29, CD90, CD44 and CD45 were respectively (99.80±0.19)%, (99.62±0.24)%, (96.86±1.27)%, (0.82±0.06)%, while after transfection the positive rates were (99.59±0.34)%, (98.06±1.27)%, (95.23±0.71)%, (10.23±0.59)%, representing no sig-nificant difference before and after PDYN transfection. Lentiviral vector of PCDH-CMV-PDYN-EF1-copGFP was successfully construc-ted after the identification of PCR amplification, cloning and sequencing. The titer of recombined lentiviruses was 5×106IU/mL. The best MOI of lentiviruses was 100 according to the results of GFP and western blot. Western blot and qPCR suggested PDYN gene signif-icantly increased in BM-MSCs after lentiviral transfection ( P<0.05) , and immunohistochemical staining indicated DYN protein also in-creased greatly. Conclusion BM-MSCs are successfully cultured and the overexpressed rat PDYN gene lentivirus vector is also suc-cessfully constructed;PDYN gene is highly and stably expressed and DYN protein is secreted in BM-MSCs.

Objective:To evaluate the role of spinal cord mitochondrial autophagy in alleviation of diabetic neuropathic pain (DNP) by curcumin in mice.Methods:SPF healthy male C57BL/6 mice, aged 2 months, weighing 20-25 g, in which DNP model was established by intraperitoneal injection of streptozotocin (STZ) 130 mg/kg, were used in this study.A total of 36 mice with successfully established DNP model were divided into 3 groups ( n=12 each) using the random number table method: DNP group, DNP + curcumin group (DPR group), and DNP + curcumin + cyclosporine A group (DRC group). Another 12 C57BL/6 mice were selected and served as normal control group (NC group), and the equal volume of normal saline was intraperitoneally injected.In group DPR, curcumin 200 mg/kg was administered by intragastric gavage, once a day, for 7 consecutive days.In group DRC, the mitochondrial autophagy inhibitor cyclosporine A 10 mg/kg was intrathecally injected once a day for 7 consecutive days before each administration of curcumin.The equal volume of normal saline was administered by intragastric gavage at the same time point, once a day, for 7 consecutive days in group NC and group DNP.The mechanical paw withdrawal threshold (MWT) was measured before intragastric gavage and at 1, 3, 5 and 7 days after intragastric gavage.After the last behavioral testing, the L 4-6 spinal cord tissues were removed for determination of the mitochondrial membrane potential and ROS content (by JC-1 and DCFH-DA combined with flow cytometry), expression of microtubule-associated protein light chain 3 (LC3), Beclin1 and P62 (by Western blot), and mitochondrial autophagosomes (by transmission electron microscopy) and for microscopic examination of the co-expression of LC3-Ⅱwith mitochondrial translocase outer membrane protein 20 (TOM20) (using immunofluorescence double-labeling technique). Results:Compared with group NC, the MWT and mitochondrial membrane potential were significantly decreased, the ROS content and LC3-Ⅱ/Ⅰ ratio were increased, the expression of Beclin1 was up-regulated, the expression of P62 was down-regulated ( P<0.05), the number of mitophagosomes developed was increased, and the co-expression of LC3-Ⅱwith TOM20 was increased in group DNP.Compared with group DNP, the MWT and mitochondrial membrane potential were significantly increased, the ROS content was decreased, and LC3-Ⅱ/Ⅰ ratio was increased, the expression of Beclin1 was up-regulated, the expression of P62 was down-regulated ( P<0.05), the number of mitophagosomes developed was increased, and the co-expression of LC3-Ⅱwith TOM20 was increased in group DPR.Compared with group DPR, the MWT and mitochondrial membrane potential were significantly decreased, the ROS content was increased, LC3-Ⅱ/Ⅰ ratio was decreased, the expression of Beclin1 was down-regulated, the expression of P62 was up-regulated ( P<0.05), the number of mitophagosomes developed was decreased, and the co-expression of LC3-Ⅱ with TOM20 was decreased in group DRC. Conclusions:The mechanism by which curcumin reduces DNP may be related to the up-regulation of mitochondrial autophagy in the spinal cord and improvement in mitochondrial function in mice.

Objective:To evaluate the role of lactate-induced mitochondrial division of spinal cord neurons in diabetic neuropathic pain (DNP) in mice.Methods:Thirty-six SPF-grade healthy adult male C57BL/6 mice, aged 2 months, weighing 20-25 g, were divided into 3 groups ( n=12 each) using a random number table method: control group (CON group), DNP group, and DNP+ sodium oxalate group (OXA group). The diabetic model was established by intraperitoneal injection of streptozotocin 130 mg/kg. After the diabetic model was successfully established, sodium oxalate 750 mg/kg was intraperitoneally injected once a day for 4 consecutive weeks to inhibit lactate production in OXA group, while the equal volume of normal saline was given instead at the same time in C group and DNP group. The mechanical paw withdrawal threshold (MWT) of the left hindpaw was measured before developing the model and at 1, 2, 3 and 4 weeks after developing the model. After completing the last behavioral testing, the spinal cord tissue of the lumbar segment (L 4-6) was taken for determination of the levels of lactate in serum and spinal cord tissues (by the colorimetric method), expression of the mitochondrial membrane potential, reactive oxygen species (ROS) content (using JC-1 or DHE probes), expression of mitochondrial dynamin-related protein 1 (Drp1) and mitochondrial protein mitofusin 2 (Mfn2) (by Western blot), and co-expression of Drp1 and neuronal neuronal marker neuronal nuclear protein (NeuN) (by immunofluorescence double labeling) and for examination of the structure and the number of mitochondria (with a transmission electron microscope). Results:Compared with C group, the MWT was significantly decreased after developing the model, the levels of lactate in serum and spinal cord tissues and ROS content in the spinal cord were increased, the mitochondrial membrane potential was decreased, the Drp1 expression was up-regulated, the Mfn2 expression was down-regulated, the number of mitochondria was increased, the area was reduced ( P<0.05), and the co-expression of Drp 1 and NeuN was increased in DNP group and OXA group. Compared with DNP group, the MWT was significantly increased after developing the model, the levels of lactate in serum and spinal cord tissues and ROS content in the spinal cord were decreased, the mitochondrial membrane potential was increased, the Drp1 expression was down-regulated, the Mfn2 expression was up-regulated, the number of mitochondria was decreased, the area was increased ( P<0.05), and the co-expression of Drp 1 and NeuN was decreased in OXA group. Conclusions:Lactate-induced excessive mitochondrial division of spinal cord neurons can lead to mitochondrial dysfunction, which may be involved in the maintenance mechanism of DNP in mice.