The Effect of Melatonin on Radicular Pain in a Rat Model of Lumbar Disc Herniation.

STUDY DESIGN: Controlled, randomized, animal study. OBJECTIVE: To investigate the effect of melatonin and its receptors on radicular pain and the possible mechanisms. SUMMARY OF BACKGROUND DATA: Lumbar disc herniation (LDH) may induce radicular pain, but the mechanism is not clear and therapeutic effect is still poor. Previously we report central sensitization meaning potentiation of spinal nociceptive synaptic transmission is the critical cause of radicular pain. Melatonin (Mel) has been reported to promote hippocampal synaptic transmission and thus improve learning ability. But the effect of Mel on spinal synaptic transmission and radicular pain are not clear. METHODS: Rat LDH model was induced by autologous nucleus pulposus (NP) implantation. Melatonin was delivered intraperitoneally four times a day, from day 1 to day 3 after surgery. Melatonin receptor agonist and antagonists were delivered intrathecally for 3 days as well. Mechanical and thermal pain thresholds were assessed by von Frey filaments and hotplate test respectively. Electrophysiological recording was employed for survey C-fiber evoked field potentials. The protein level of N- methyl-D-aspartate submit 2A (NR2A), NR2B, melatonin receptor 1 (MT1), and receptor 2 (MT2) was evaluated by western blotting. Spinal expression of calcitonin gene related peptides (CGRP), isolectin b4 (IB4), and neurofilament-200 (NF200) was displayed by immunofluorescence staining. RESULTS: Melatonin significantly increased mechanical and thermal pain thresholds, lasting at least to day 5 after surgery. Melatonin decreased C-fiber evoked field potentials; decreased spinal NR2B protein level; reduced spinal CGRP, and IB4 expression. MT2 was upregulated after NP implantation and was co-localized with neuron and microglia. MT2 receptor agonist simulated the effect of Mel, and both MT receptor broadspectrum antagonist and MT2 specific antagonist abolished the effect of MT2 receptor agonist. CONCLUSION: Melatonin alleviates radicular pain from LDH by inhibiting central sensitization via binding with its receptor 2, decreasing spinal CGRP, IB4, and NR2B expression.

[Dynamic changes of the PGAM1 expression in the mouse testis exposed to single heat stress].

OBJECTIVE: To investigate the expression of phosphoglycerate mutase 1 (PGAM1) in the mouse testis after exposure to single heat stress (SHS). METHODS: We randomly assigned 32 C57 male mice to an SHS (n = 16) and a control group (n = 16), the former bathed in water at 43 ℃ and the latter at 25 ℃ for 15 minutes. At 1 and 7 days after exposure, we harvested the testicular tissue for observation of the morphological changes of testicular cells by HE staining and determination of the location and expression of the PGAM1 protein by immunohistochemistry and Western blot. RESULTS: The testis volume of the mice were reduced significantly, the spermatogenic tubules were disorganized, and the cells were reduced in number after heat stress and basically disappeared after 7 days. Immunohistochemistry showed extensive expression of the PGAM1 protein in the testicular spermatogenic tubules of the SHS-exposed mice, significantly higher than in the control group at 1 day after exposure, which was down-regulated in the testis tissue at 7 days, but still markedly higher than that in the control. Western blot exhibited significantly up-regulated expression of the PGAM1 protein after heat stress compared with that in the control group. CONCLUSIONS: The expression of the PGAM1 protein undergoes dynamic changes in the mouse testis after exposed to single heat stress, which is related to heat stress-induced proliferation and division of testicular spermatogenic cells.

PGAM1 knockdown is associated with busulfan­induced hypospermatogenesis and spermatogenic cell apoptosis.

Phosphoglycerate mutase 1 (PGAM1) is reported to be involved in spermatogenic dysfunction. However, the association between PGAM1 and busulfan­induced hypospermatogenesis and spermatogenic cell apoptosis remains unclear. The aim of the current study was to investigate the association between PGAM1 expression and busulfan­induced hypospermatogenesis, and the effect of PGAM1 expression on spermatogenic cell apoptosis. PGAM1 expression was detected in mouse models of busulfan­induced hypospermatogenesis by western blotting, reverse transcription­quantitative polymerase chain reaction and immunohistochemistry. Then, spermatogenic cell apoptosis in mouse models of busulfan­induced hypospermatogenesis was assessed by TUNEL assay. The effect and potential mechanism of PGAM1 downregulation on spermatogenic cells were further investigated. The results indicated that PGAM1 expression was significantly downregulated in the mouse models of busulfan­induced hypospermatogenesis, compared with those with normal spermatogenesis (P<0.05). Furthermore, the TUNEL assay revealed that the apoptosis of spermatogenic cells was accelerated in the mouse model of busulfan­induced hypospermatogenesis. In addition, PGAM1 knockdown promoted the apoptosis of spermatogenic cells in vitro, which was associated with the P53/Caspase 3/Caspase 6/Caspase 9 signaling pathway. In conclusion, these data indicate that PGAM1 knockdown is associated with busulfan­induced hypospermatogenesis and contributes to spermatogenic cell apoptosis by regulating the P53/Caspase 3/Caspase 6/Caspase 9 signaling pathway.

Puerarin alleviate radicular pain from lumbar disc herniation by inhibiting ERK-dependent spinal microglia activation.

Lumbar disc herniation is a common cause of radicular pain, but the mechanism remains ambiguous and the treatment stays unsatisfied. Many studies revealed a traditional Chinese medicine puerarin may moderate chronic pain from diabetes and nerve injury. Thus far, the role and mechanism of puerarin in radicular pain is still unknown. In this study, by using a rat model of lumbar disc herniation, which was induced by autologous nucleus pulposus (NP) implantation, the analgesic effect of puerarin on radicular pain was tested. Puerarin was delivered intraperitoneally form 1 h before surgery, and once daily for 7 days. The results demonstrated that NP implantation induced long-lasting pain, characterized by decrease of paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) in ipsilateral hindpaws, as long as day 20 after surgery. Spinal phosphorylated extracellular signal-regulated kinase (p-ERK) was up-regulated from day 5 to day 20 after surgery in ipsilateral but not contralateral side, and p-ERK was mainly co-localized with microglia. Puerarin decreased p-ERK expression from day 7 to day 20 after surgery. Puerarin or ERK inhibitor PD98059 alleviated pain behaviors, decreased expression of microglia marker ionized calcium-binding adaptor molecule 1 (Iba-1) in rats with NP implantation. The results suggested puerarin may alleviate radicular pain by inhibiting ERK-dependent or accompanied spinal microglia activation.

Src-family kinases activation in spinal microglia contributes to central sensitization and chronic pain after lumbar disc herniation.

Background Lumbar disc herniation is a major cause of radicular pain, but the underlying mechanisms remain largely unknown. Spinal activation of src-family kinases are involved in the development of chronic pain from nerve injury, inflammation, and cancer. In the present study, the role of src-family kinases activation in lumbar disc herniation-induced radicular pain was investigated. Results Lumbar disc herniation was induced by implantation of autologous nucleus pulposus, harvest from tail, in lumbar 4/5 spinal nerve roots of rat. Behavior test and electrophysiologic data showed that nucleus pulposus implantation induced persistent mechanical allodynia and thermal hyperalgesia and increased efficiency of synaptic transmission in spinal dorsal horn which underlies central sensitization of pain sensation. Western blotting and immunohistochemistry staining revealed that the expression of phosphorylated src-family kinases was upregulated mainly in spinal microglia of rats with nucleus pulposus. Intrathecal delivery of src-family kinases inhibitor PP2 alleviated pain behaviors, decreased efficiency of spinal synaptic transmission, and reduced phosphorylated src-family kinases expression. Furthermore, we found that the expression of ionized calcium-binding adapter molecule 1 (marker of microglia), tumor necrosis factor-α, interleukin 1 -ß in spinal dorsal horn was increased in rats with nucleus pulposus. Therapeutic effect of PP2 may be related to its capacity in reducing the expression of these factors. Conclusions These findings suggested that central sensitization was involved in radicular pain from lumbar disc herniation; src-family kinases-mediated inflammatory response may be responsible for central sensitization and chronic pain after lumbar disc herniation.

PGAM1 is Involved in Spermatogenic Dysfunction and Affects Cell Proliferation, Apoptosis, and Migration.

OBJECTIVE: To investigate the correlation between PGAM1 and spermatogenic dysfunction and to evaluate the effect of expression of PGAM1 on the function of germ cells. METHODS: Expression of PGAM1 was detected in 40 cases of infertile males with definite pathological diagnosis and 12 cases of mouse models with spermatogenic dysfunction by immunohistochemistry. Then, cell proliferation, apoptosis, and migration were evaluated when expression of PGAM1 was knocked down by a specific small interfering RNA in GC1 and TM4 cells. RESULTS: The positive rates of PGAM1 in patients with normal spermatogenesis, mild hypospermatogenesis, severe hypospermatogenesis, and Sertoli cell-only syndrome were 90%, 80%, 10%, 100%, respectively, and the difference was significant (P < .001). Meanwhile, expression of PGAM1 was found to be significantly decreased in mouse models with spermatogenic dysfunction. Moreover, when expression of PGAM1 was knocked down in GC1 cells, the proliferation and migration were significantly inhibited, but the rate of apoptosis was significantly increased. Furthermore, PGAM1 downregulation in TM4 cells significantly inhibited proliferation and promoted apoptosis but didn't affect migration. CONCLUSION: PGAM1 correlates with spermatogenic distinction and affects the function of cell proliferation, apoptosis and migration.