Objective:To evaluate the role of spinal cord neuron Src-associated-in-mitosis-68-kDa (SAM68)-transient receptor potential vanilloid-1 channel (TRPV1) signaling pathway in diabetic neuropathic pain (DNP) in mice.Methods:Forty SPF male C57BL/6 mice, aged 8 weeks, weighing 18-22 g, were used in this study. Diabetes mellitus was induced by intraperitoneal streptozotocin 0.12 mg/g, and successful DNP model was defined as decrease in the mechanical paw withdrawal threshold (MWT) of the hind limb≥50% of the baseline value. Twenty-four mice with DNP at 4 weeks after developing the model were divided into 3 groups ( n=8 each) using a simple random sampling: DNP group, SAM68 knocked down group (KD group) and virus control group (VC group). Eight diabetic mice with decrease in MWT <50% were randomly selected as non-DNP group (ND group), and 8 normal mice were randomly selected as control group (NC group). At 4 weeks after developing the diabetes mellitus model, SAM68 gene silencing virus and empty virus were injected into the lumbar enlargement of the spinal cord in KD group and VC group, respectively. MWT was measured before developing the diabetes mellitus model and at 4 and 6 weeks after developing the diabetes mellitus model. The mice were sacrificed after the end of MWT measurement at week 6 after developing the model, spinal cord tissues were collected and the expression of SAM68 and TRPV1 was detected by Western blot, and their localization in the spinal cord was observed by immunofluorescence. Results:Compared with NC and ND groups, the MWT was significantly decreased at 4 and 6 weeks after developing the model, and the expression of SAM68 and TRPV1 in spinal cord tissues was up-regulated in DNP group ( P<0.05). Compared with DNP group, the MWT was significantly increased at 6 weeks after developing the model, the expression of SAM68 and TRPV1 in spinal cord tissues was down-regulated, and no significant change was found in the parameters mentioned above in VC group ( P>0.05). SAM68 and TRPV1 were expressed in neurons in the same region of the spinal cord. Conclusions:Activation of SAM68-TRPV1 signaling pathway in spinal cord neurons is involved in the pathophysiological mechanism of DNP 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.

Objective:To evaluate the effect of intrathecal insulin-like growth factor-1 (IGF-1) on chemotherapy-induced neuropathic pain (NP) in mice.Methods:Forty clean-grade healthy male C57 mice, aged 7-9 weeks, weighing 22-24 g, were divided into 4 groups ( n=10 each) using a random number table method: control group (group C), chemotherapy-induced NP group (group CIPN), low-dose IGF-1 group (group I1) and high-dose IGF-1 group (group I2). In CIPN, I1 and I2 groups, oxaliplatin 5 mg/kg was intraperitoneally injected for 5 consecutive days to establish chemotherapy-induced NP model.Normal saline 0.2 ml was given in group C. After measurement of the pain threshold at 10 days after establishment of the model, IGF-1 0.5 and 1.0 μg were intrathecally injected in group I1 and group I2, respectively.Normal saline 5 μl was intrathecally injected in C and CINP groups.Mechanical withdrawal threshold (MWT) was measured at 3, 5, 8, 10, 11, 13 and 15 days after establishment of the model.After measurement of the pain threshold at 15 days after establishment of the model, the expression of spinal IGF-1, IGF-1receptor (IGF-1R), interleukin (IL)-17A, IL-1β and tumor necrosis factor (TNF)-α was detected, and IGF-1 positive cells were counted using immunofluorescence. Results:Compared with group C, MWT was significantly decreased, the expression of spinal IGF-1 was down-regulated, the count of IGF-1 positive cells was decreased, and expression of IL-17A, IL-1β and TNF-α was up-regulated at 3-25 days after establishment of the model in CINP, I1 and I2 groups ( P<0.05). Compared with group CIPN, MWT was significantly increased at 15 days after establishment of the model in group I1, and MWT was increased, the expression of spinal IGF-1 was up-regulated, the count of IGF-1 positive cells was increased, and expression of IL-17A, IL-1β and TNF-α was down-regulated at 13 and 15 days after establishment of the model in group I2 ( P<0.05). Compared with group I1, the count of IGF-1 positive cells in spinal dorsal horn was increased in group I2 ( P<0.05). There was no significant difference in the expression of spinal IGF-1R among the 4 groups ( P>0.05). Conclusion:Intrathecal IGF-1 can alleviate chemotherapy-induced NP, and the mechanism may be related to inhibiting the inflammatory responses in spinal cord of mice.

Objective To evaluate the role of spinal cord tumor necrosis factor receptor-associated factor 6 (TRAF6)/nuclear factor kappa B (NF-κB)signaling pathway in the development of diabetic neuropathic pain (DNP)in rats.Methods Clean-grade healthy adult male Sprague-Dawley rats,aged 2 months,weighing 180-230 g,in which IT catheters were implanted,were used in this study.Streptozotocin 60 mg/kg was intraperitoneally injected after IT catheterization to establish the model of DNP.Twelve DNP rats were divided into 2 groups (n =6 each) by a random number table method:DNP group and DNP plus TRAF6 inhibitor group (group DTR).Another 6 age-matched Sprague-Dawley rats were used as normal control group (group NC).The rats in group DC and group DTR received IT injection of dimethyl sulfoxide 10 μl and TRAF6 inhibition 10 μg,respectively,once a day for 7 consecutive days starting from day 21 after establishing the model.The mechanical paw withdrawal threshold (MWT) was determined before establishing the model (T1),on 7,14 and 21 days after establishing the model (T2-4),and on 1,4 and 7 days after IT injection (T5-7).The rats were sacrificed after the last MWT measurement,and the L3-5 segments of the spinal cord were removed for determination of the expression of TRAF6 and NFκB p65 by Western blot.Results Compared with group NC,the MWT at T3-7 in group DC and at T3-6 in group DTR was significantly decreased,and the expression of spinal TRAF6 and NF-κB p65 was up-regulated in DC and DTR groups (P＜0.05).Compared with group DC,the MWT was significantly increased at T6-7,and the expression of spinal TRAF6 and NF-κB p65 was down-regulated in group DTR (P ＜ 0.05).Conclusion Spinal cord TRAF6/NF-κB signaling pathway is involved in the development of DNP in rats.

Objective To evaluate the effect of pulsed radiofrequency (PRF) on the phenotypic transformation of the lumbar sympathetic ganglion (LSG) in the rats with diabetic neuropathic pain (PDN).Methods Twenty-four clean-grade healthy adult male Sprague-Dawley rats,aged 2 months,weighing 180-220 g,were divided into 4 groups (n =6 each) according to the method of random number table:control group (group C),group PDN,group PRF,and PRF control group (group PC).The PDN model was established by intraperitoneal injection of streptozotocin 60 mg/kg in anesthetized rats.Citrate-sodium citrate buffer 6 ml/kg was intraperitoneally injected in group C.Group PC only received radiofrequency needle puncture.PRF was performed on the right L3 LSG in group PRF.The mechanical paw withdrawal threshold (MWT) to yon Frey filament stimulation was measured before intraperitoneal injection (baseline,T0),before PRF and at 1,3,5,7 and 14 days after PRF.The rats were then sacrificed,and ipsilateral L3 LSGs were removed for determination of the expression of tyrosine hydroxylase (TH) and vesicle glutamate transporter2 (VGLUT2) in LSGs (by double immunofluorescent staining) and for examination of pathological changes (with a light microscope).The number of neurons expressing VGLUT2 was counted.Results Compared with group C,the MWT was significantly decreased at T1-6,and the number of neurons expressing VGLUT2 was increased at T6 in PDN,PC and PRF groups (P＜0.05).Compared with PDN and PC groups,the MWT was significantly increased at T2-6,and the number of neurons expressing VGLUT2 was decreased at T6 in group PRF (P＜0.05).TH expression in LSGs was found,and no VGLUT2 expression in LSGs was observed in group C,the expression of TH and VGLUT2 in LSGs was found in the other three groups,especially in PDN and PC groups,and most of the neurons expressing VGLUT2 expressed TH simultaneously.Conclusion The mechanism by which PRF mitigates PDN is related to inhibiting the phenotypic transformation of LSGs in the rats.

Objective To evaluate the role of microRNA9 (miR-9) in spinal dorsal horn neurons in over-expression of calcium homeostasis modulator 1 (CALHM1) in rats with diabetic neuropathic pain (DNP).Methods Ninety-three healthy male Sprague-Dawley rats,aged 2 months,weighing 180-200 g,were used in the study.Diabetes mellitus was induced by intraperitoneal 1％ streptozocin (STZ) 60 mg/kg.The experiment was performed in two parts.Experiment Ⅰ The rats were divided into control group (group C,n=10) and DNP group (n=83) using a random number table.The mechanical paw withdrawal threshold (MWT) was measured before STZ injection and at 1,2,3,4,5 and 6 weeks after STZ injection.The expression of miR-9 in the spinal dorsal horn was determined using the in situ hybridization at 6 weeks after STZ injection.Experiment Ⅱ The rats with DNP were selected at 6 weeks after STZ injection,and the spinal dorsal horn neurons were isolated and cultured.The neurons were seeded in culture plates at the density of 5×106 cells/ml (2 ml/well) and divided into 2 groups (n=18 each) using a random number table:control group (group C) and miR-9 antisense oligonucleotide group (group ASO).The neurons were cultured in normal culture atmosphere in group C.In group ASO,the single nucleotide sequence of miR-9 antisense oligonucleotide sequence 5'-UUCUCCGAACGUGUCACGUTT-3'was added with the final concentration of 100 pmol/L.The expression of miR-9 and CALHM1 mRNA was detected using quantitative real-time polymerase chain reaction at 48 h of incubation.The expression of CALHM1 was detected by Western blot at 72 h of incubation.Results Experiment Ⅰ Compared with group C,the MWT was significantly decreased at 2-6 weeks after STZ injection,and the expression of miR-9 in the spinal dorsal horn was up-regulated in group DNP (P＜0.05).Experiment Ⅱ Compared with group C,the expression of miR-9 and CALHM1 protein and mRNA in spinal dorsal horn neurons was significantly down-regulated in group ASO (P＜0.05).Conclusion miR-9 in spinal dorsal horn neurons probably mediates the over-expression of CALHM1 in rats with DNP.

Objective To evaluate the effect of intrathecal cardamonin on diabetic neuropathic pain (DNP) in rats.Methods Healthy adult male Sprague-Dawley rats,aged 2 months,weighing 180-220 g,were included in the study.Diabetes mellitus was produced by intraperitoneal injection of streptozotocin 60 mg/kg after intrathecal catheterization.When decrease in pain threshold＞50％ of the baseline at 21 days after diabetes mellitus was produced,the induction of DNP was considered successful.Twenty-four rats with DNP were divided into 4 groups (n=6 each) using a random number table:group DNP,rapamycin group (group R),cardamonin 50 μg group (group D50) and cardamonin 100 μg group (group D100).Another 6 healthy age-matched rats were used as normal control (group C).In DNP,R,D50 and D100 groups,dimethyl sulfoxide 10 μl,rapamycin 5 μg and cardamonin 50 μg and 100 μg were intrathecally injected,respectively,once a day for 7 consecutive days starting from 21 days after successful estabiishment of the model.Mechanical paw withdrawal threshold (MWT) was measured before establishment of the model,on 7,14 and 21 days after establishment of the model,and on 1,4 and 7 days after intrathecal injection.The rats were sacrificed after the last MWT measurement,and their L3-5 segments of the spinal cord were removed for determination of the expression of S6K,p-S6K and synapsin Ⅱ by Western blot.Results Compared with group C,MWT was significantly decreased,and the expression of spinal S6K,p-S6K and synapsin Ⅱ was up-regulated in group DNP (P＜0.05 or 0.01).Compared with group DNP,MWT was significantly increased,and the expression of spinal S6K,p-S6K and synapsin Ⅱ was down-regulated in R,D50 and D100 groups (P＜0.05 or 0.01).Compared with group D50,MWT was significantly increased,and the expression of spinal S6K,p-S6K and synapsin Ⅱ was down-regulated in group D100 (P＜0.05).Conclusion Intrathecal cardamonin can relieve DNP in rats,and the mechanism is related to inhibiting spinal mammalian target of rapamycin activity and down-regulating the expression of synapsin Ⅱ.

Objective To investigate the role of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) protein in the spinal cord neurons in diabetic neuropathic pain (DNP) in rats.Methods Male Sprague-Dawley rats,aged 2 months,weighing 180-200 g,were studied.Diabetes mellitus was induced by single intraperitoneal injection of streptozotocin (STZ) 60 mg/kg.Sixteen rats with DNP were randomly divided into 2 groups (n =8 each) using a random number table:DNP group and DNP+PTEN inhibitor bpv (pic) group (DPN-bpv group).Another 16 rats were equally and randomly divided into either control group (group C) or bpv group.In DNP-bpv and bpv groups,bpv (pic) 0.2mg/kg was injected intraperitoneally once a day within 14-28 days after injection of STZ.Before STZ injection (T1),and at 2,7,14,21 and 28 days after STZ injection (T2-6),the mechanical paw withdrawal threshold (MWT) was measured.After measurement of MWT,the rats were sacrificed,and the lumbar segments of the spinal cord (L4.5) were removed for determination of PTEN protein activity (by ELISA) and Akt (s473) phosphorylation (by Western blot).Results Compared with group C,the MWT was significantly decreased at T4-6,and the PTEN protein activity and Akt (s473) phosphorylation were significantly increased in DNP and DNP-bpv groups (P＜0.05 or 0.01).Compared with group DNP,the MWT was significantly increased at T6,and the PTEN protein activity and Akt (s473) phosphorylation were significantly decreased in group DNP-bpv (P＜0.05).Conclusion PTEN protein in the spinal cord neurons is involved in the maintenance of DNP in rats.

Objective To compare the anesthetic efficacy of ketamine and sevoflurane for foreskin ligation in the pediatric patients.Methods A total of 120 pediatric patients,aged 2-6 yr,weighing 10-18 kg,scheduled for elective foreskin ligation,were equally and randomly divided into ketamine group (group K) and sevoflurance group (group S).In group K,atropine 0.25 mg/kg and ketamine 2 mg/kgwere injected intravenously,and foreskin ligation was performed after loss of eyelash reflex.In group S,8％ sevoflurance was inhaled using the tidal volume technique,the concentration inhaled was adjusted to 4％ after loss of eyelash reflex,and then foreskin ligation was performed.The occurrence of crying before and during anesthesia induction,induction time,emergence time,occurrence of agitation during emergence from anesthesia and duration of agitation were recorded.Results Compared with group K,the rate of crying was significantly decreased,the emergence time was shortened (P＜0.05),and no significant difference was found in the induction time,incidence of agitation during emergence from anesthesia,and duration of agitation in group S (P＞0.05).Conclusion Sevoflurance provides better anesthetic efficacy than ketamine when applied for foreskin ligation in the pediatric patients.

Objective To investigate the effect of propofol on human renal tubule epithelial cell (HK-2 cells) fibrosis induced by ATP depletion/recovery and the role of transforming growth factor β activated kinase 1 (TAK1) in it.Methods HK-2 cells were seeded in 96-well plates,and randomly divided into 4 groups (n =36 each) using a random number table:control group (group C),ATP depletion/recovery group (group D/R),propofol group (group P),and TAK1 over-expression group (group T).HK-2 cells were exposed to antimycin A for 1 h and then returned to normal culture medium to establish the model of ATP depletion/recovery-induced injury.At 1 h before ATP depletion,the cells were incubated for 1 h in the DMEM liquid culture medium containing propofol with the final concentration of 20 μmol/L in group P,and the cells were incubated for 1 h in the DMEM liquid culture medium containing propofol with the final concentration of 20 μmol/L and TAK1 with the titer of 2× 107 TU/ml in group T,and the other treatments were similar to those previously described in group D/R.At 12 h after ATP recovery,the cell viability was evaluated by methyl thiazolyl tetrazolium assay,and cell apoptosis was detected using TUNEL and scored.The expression of TAK1 was detected using Western blot at 12,24 and 48 h after ATP recovery.The expression of α-smooth muscle actin (αSMA),fibronectin (FN),and collagen protein 1 (COL1) was measured at 48 h after ATP recovery.Results Compared with group C,the cell viability was significantly decreased,the apoptosis score was increased,and the expression of TAK1,COL1,αSMA and FN was up-regulated after ATP recovery in D/R,P and T groups (P＜0.05).Compared with group D/R,the cell viability was significantly increased,the apoptosis score was decreased,and the expression of TAK1,COL1,αSMA and FN was down-regulated after ATP recovery in P and T groups (P＜0.05).Compared with group P,the cell viability was significantly decreased,the apoptosis score was increased,and the expression of TAK1,COL1,αSMA and FN was up-regulated after ATP recovery in group T (P＜ 0.05).Conclusion Propofol can reduce HK-2 cell fibrosis induced by ATP depletion/recovery,and the mechanism may be related to down-regulation of TAK1 expression.