Effect of osthole on p38 MAPK signaling related pathway of rat model with inflammatory radicular pain induce by nucleus pulposus in the spinal horn / 中国药理学通报

Aim To observe the effect of epidurally application of osthole on the model of nucleus pulposusinduced inflammatory radicular pain and the expression of p38 MAPK signaling related pathway in the spinal dorsal horn of rats.Methods The model of radicular pain was generated by putting nucleus pulposus to the L5 dorsal root ganglion (DRG).50％ MWT was measured using Von Frey filaments to calculate mechanical pain threshold before and after operation.50 μL of 20 g · L-1 osthole was administered epidurally in group Ost and 50 μL of 100 mL · L-1 DMSO in group DMSO at postoperative day (POD).The expression of phosphorylated p38 (p-p38),IL-18 and IL-18R in the lumbar spinal dorsal horn was detected by Western blot.IL-18 mRNA was assessed by real-time PCR.Results The mechanical pain threshold significantly decreased after operation (P ＜ 0.05),while the expression of protein p-p38 MAPK,IL-18,IL-18R and IL-18 mRNA was significantly different.Compared with DMSO group,50％ MWT was significantly increased and accompanied with the decrease of protein p-p38,IL-18,IL-lgR and IL-18 mRNA in Ost group after drug administration (P ＜ 0.05).The correlation analysis between protein concentration of p38 MAPK and IL-18 mRNA showed that the Spearman correlation coefficient was 0.9 (P ＜ 0.05).Conclusion p-p38 and IL-18 of spinal dorsal horn participate in the rat model with inflammatory radicular pain induced by nucleus pulposus,and IL-18R plays a role in maintenance of the pain.Osthole administered epidurally in the early stage of pain could alleviate the pain for a long time,which may be related with inhibiting p38 MAPK signaling related pathways.

Autologous nucleus pulposus transplantation to lumbar 5 dorsal root ganglion after epineurium dissection in rats: a modified model of non-compressive lumbar herniated intervertebral disc / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Nucleus pulposus of intervertebral discs has proinflammatory characteristics that play a key role in neuropathic pain in lumbar herniated intervertebral disc. One of the most commonly used animal models (the traditional model) of non-compressive lumbar herniated intervertebral disc is created by L4-L5 hemilaminectomy and the application of autologous nucleus pulposus to cover the left L4 and L5 nerve roots in rats. However, such procedures have the disadvantages of excessive trauma and low success rate. We proposed a modified model of non-compressive lumbar herniated intervertebral disc in which only the left L5 dorsal root ganglion is exposed and transplanted with autologous nucleus pulposus following incision of epineurium. We aimed to compare the modified model with the traditional one with regard to trauma and success rate.</p><p><b>METHODS</b>Thirty Sprague-Dawley male rats were randomized into three groups: sham operation group (n = 6), traditional group (n = 12), and modified group (n = 12). The amount of blood loss and operative time for each group were analyzed. The paw withdrawal threshold of the left hind limb to mechanical stimuli and paw withdrawal latency to heat stimuli were examined from the day before surgery to day 35 after surgery.</p><p><b>RESULTS</b>Compared with the traditional group, the modified group had shorter operative time, smaller amount of blood loss, and higher success rate (91.7% versus 58.3%, P < 0.05). There was no decrease in paw withdrawal latency in any group. The sham operation group had no decrease in postoperative paw withdrawal threshold, whereas the modified and traditional groups had significant reduction in paw withdrawal threshold after surgery (mechanical hyperalgesia).</p><p><b>CONCLUSIONS</b>Transplantation of nucleus pulposus onto the L5 dorsal root ganglion following incision of epineurium in rats established an improved animal model of non-compressive lumbar herniated intervertebral disc with less trauma and more stable pain ethology.</p>

Analysis of factors leading to prescheduled analgesic withdrawal in patients with postoperative epidural analgesia / 南方医科大学学报

<p><b>OBJECTIVE</b>To analyze the factors leading to prescheduled analgesic withdrawal in patients with postoperative epidural analgesia.</p><p><b>METHODS</b>A retrospective study of 4876 patients with postoperative epidural analgesia was conducted and the effect of analgesia and incidence of prescheduled analgesic withdrawal were recorded. The factors precipitating the occurrences of analgesic withdrawal and complications were analyzed.</p><p><b>RESULTS</b>Early analgesic withdrawal occurred in 113 cases (2.3%), among which 74 (0.5%) were due to factors irrelevant to analgesic complications. Analgesia-related complications occurred in 578 patients, but only 39 (0.7%) of them needed discontinuation of the analgesics.</p><p><b>CONCLUSION</b>Prescheduled analgesic withdrawal is predominantly due to technical inadequacies rather than complications arising from the analgesics, and improvement of the operation skills for postoperative analgesia may reduce early analgesia discontinuation and enhance the patients' satisfaction.</p>

Antisense hTERT inhibits gene expression and functional activity of telomerase in leukemia cell lines / 中华病理学杂志

<p><b>OBJECTIVE</b>To explore the inhibitory effects of gene expression and functional activity of telomerase in leukemia cell lines by in vitro antisense hTERT treatment.</p><p><b>METHODS</b>An antisense hTERT eukaryotic expression vector was constructed by using gene recombination technique, targeting the 5' end mRNA sequence of the telomerase catalytic subunit. The vector expression in leukemia cell lines (HL60 and K562) was achieved by transfection using the SuperFect transfection reagent (Qiagen). After transfection, ectopic expression of the telomerase catalytic subunit was analyzed by quantitative fluorescence real-time RT-PCR, and cellular apoptosis and cell cycle parameters were evaluated by flow cytometry respectively.</p><p><b>RESULTS</b>An antisense pcDNA-hTERT eukaryotic expression vector was successfully constructed. Leukemia cell lines transfected with antisense hTERT constructed displayed a significant inhibition of gene expression of telomerase and its activity in vitro, as compared with the result of the control groups (without transfection and vector control).</p><p><b>CONCLUSION</b>In-vitro antisense hTERT expression may down-regulate the gene expression and biological activity of telomerase in leukemia cells, suggesting a possibility of gene therapy against human malignancy through the telomerase-targeted molecular mechanism.</p>

Effects of antisense human telomerase reverse-transcript protein subunit (hTERT) gene on biological characteristics of hepatoblastoma cell line in vitro / 中华儿科杂志

<p><b>OBJECTIVE</b>Telomerase, a complex of ribose and nucleoprotein, is a specific marker of tumor, which expresses in 98% infinite cell lines and 90% malignant tumor organizations and whose function is to maintain the length of telomere. Human telomerase reverse-transcript protein subunit (hTERT) is the key element and rate-limiting factor of telomerase activity. Our study was to investigate the effects of antisense hTERT gene on biological characteristics of hepatoblastoma cell line in vitro.</p><p><b>METHODS</b>The sense and antisense hTERT eukaryotic expression vectors that we had constructed before were transfected into hepatoblastoma cell line HepG2 by using the SuperFect transfection reagent (Qiagen) according to the manufacturer's instructions, then the HepG2-s and HepG2-as of G418-resistant colonies were obtained with G418 and identified for the presence of hTERT insert by PCR with T7 and pcDNA3.1/BGH reverse primers. After that, we have detected the endogenous hTERT mRNA expression and telomerase activity by quantitative real-time RT-PCR and TRAP-silver staining assay in cells from each group. Meanwhile, MTT cellular proliferation assay, soft agar colony formation assay and flow cytometry were employed to analyze if the proliferation capacity of liver cancer cells was affected in vitro and the tumor cells could be induced to apoptosis by antisense hTERT.</p><p><b>RESULTS</b>Antisense hTERT significantly down-regulated the endogenous hTERT mRNA expression (15.35 +/- 1.72/HepG2-as, 43.8 +/- 2.89/HepG2-s, 45.2 +/- 3.46/HepG2) (n = 10, t = 7.61, P < 0.01) and telomerase activity in HepG2, compared to blank control and sense hTERT. After 20 passages of three group cells, a 7-day cell growth curve and the numbers (size) of soft agar colony formation showed the proliferation and the anchorage-independent growth in HepG2-as were significantly suppressed (50.6 +/- 4.8/HepG2-as, 113.52 +/- 8.15/HepG2-s, 119.12 +/- 10.82/HepG2) (n = 10, t = 4.54, P < 0.01 and n = 10, t = 3.96, P < 0.01), compared to HepG2 and HepG2-s. However there was a significant increase in apoptosis percentage of HepG2-as by flow cytometry (n = 10, t = 9.24, P < 0.01 and n = 10, t = 8.37, P < 0.01), compared to control group.</p><p><b>CONCLUSIONS</b>Antisense hTERT could significantly suppress the hepatoblastoma cell growth and reverse its malignant phenotypes in vitro and cause the increase in apoptosis percentage of HepG2, thus it might be applied in malignant tumor gene therapy through the telomerase-targeted molecular mechanism.</p>

The correlation between Kawasaki disease and polymorphisms of Tumor necrosis factor alpha and interleukin-10 gene promoter / 中华儿科杂志

<p><b>OBJECTIVE</b>Kawasaki disease (KD) is an acute febrile vasculitic syndrome of unknown etiology that preferentially affects coronary artery. It has been suggested that proinflammatory cytokines like tumor necrosis factor alpha (TNF-alpha) and interleukin-10 (IL-10) are key players during acute KD. Recently, the polymorphisms relative to major transcriptional start site of TNF-alpha and IL-10 gene were shown to influence the level of TNF-alpha and IL-10 production in vitro. This study was aimed to investigate the genetic association of TNF-alpha and IL-10 promoter polymorphisms in juvenile patients of Han nationality with KD, and to investigate the possible associations with clinical manifestations of the disease.</p><p><b>METHODS</b>Four polymorphism sites of TNF-alpha and IL-10 gene promoter regions from 96 children with KD were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). One hundred and sixty age-matched normal children of the Han nationality were used as control. All patients accepted Doppler echocardiography examination in order to differentiate coronary artery lesions.</p><p><b>RESULTS</b>There was significant difference in allele frequencies of -308 (A/G) site of the TNF-alpha gene between children of the Han nationality and those of Japanese and Caucasian in America. There were significant differences in the allele frequencies of -1082 (G/A), -819 (C/T) and -592 (A/C) of IL-10 gene between children of the Han nationality and their British Counterparts (P < 0.01). There was no significant difference in allele frequencies of -308 (A/G) site of TNF-alpha gene between children with KD and normal controls. There was no significant difference in the haplotypes and the allele frequencies of the above three sites of IL-10 between the two groups. However, when clinical features were examined, the genotype frequency of TNF-alpha-308A was significantly higher in IVIG-resistant KD patients than that of TNF-alpha-308G genotype (67% vs 5%, chi(c)(2) = 90.48, P < 0.01). The genotype of TNF-alpha-308A was closely associated with IVIG-resistant KD (P < 0.01, relative risk 42.25, 95% confidence interval 15.81-112.88). The haplotype frequency of IL-10 -1082A/-819T/-592A was also higher in patients with coronary artery lesion (CAL) caused by KD than those of Non-ATA haplotype (52% vs 20%, chi(2) = 18.36, P < 0.01). The haplotypes of IL-10 -1082A/-819T/-592A was significantly associated with CAL caused by KD (P < 0.01, relative risk 4.26, 95% confidence interval 2.20-8.25).</p><p><b>CONCLUSION</b>The genotype of TNF-alpha-308A is one of the important factors that probably influence the therapeutic effect of KD. The haplotypes (-1082/-819/-592) of IL-10 gene promoter might be related to the pathogenesis of coronary artery complication of KD and -1082A/-819T/-592A haplotypes might be regarded as a genetic marker of risk factor for coronary artery lesion in KD.</p>