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Aim To discuss the changes of polyamine metabolism and autophagy in aging rat heart and the effect of exogenous spermidine on autophay in aged rat heart. Methods Western blot assay was used to detect the expressions of ODC and SSAT-rate-limiting enzyme of polyamine anabolism and catabolism as well as the expression of autophagy-relevant protein LC3?/? and p62 in cardiac tissues from male Wistar rats aged 3, 6, 12 and 24 months, and the expressions of p16, LC3?/?, ATG5 and ATG7 protein were detected in cardiac tissues in rats aged 3 months, 24 months and 24 months with spermidine administrtion, respectively. The myocardial autophagosome formation was observed by transmission electron microscope. In addition, cell cross-sectional area, cell apoptosis rate and generation of ROS were evaluated. Results With heart aging in rats, ODC expression and LC3?/? ratio were degraded, and SSAT and p62 protein expression upgraded. In rats aged 24 months, myocardium showed increased p16 expression, cell cross-sectional area, cell apoptosis and ROS generation, while cell autophagosome formation decreased, p62 expression increased, the expression of LC3?/?, ATG5 and ATG7 all declined. Spermidine intervention obviously inhibited myocardial changes induced by aging, showing decrease in cell cross-sectional area, apoptosis, ROS generation and p62 expression, and increase in LC3?/?, ATG5 and ATG7 expression. Conclusions With heart aging in rats, polyamine anabolism is degraded, catabolism is upgraded, and cell autophagy declined. Exogenous spermidine might delay aging through inducing autophagy of cardiomyocytes.
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Objective To construct the pEGFP-C1-CXCL1 eukaryotic expression vector and to investigate the effect of CXCL1 on the proliferation of HepG2 cells under endoplasmic reticulum stress ( ERS).Methods Fragments of CXCL1 were obtained from the cDNA library of HepG2 cells before CXCL1 was cloned into a pEGFP-C1 vector for a recombinant plasmid pEGFP-C1-CXCL1 which was screened and identified by PCR and sequence alignment .Then,the recombinant plas-mid of pEGFP-C1-CXCL1 was transfected into human 293 T cell line and the expression of CXCL 1 was detected by fluores-cence microscopy and Western blotting.pEGFP-C1-CXCL1was furhter transfected into HepG2 cells, and CCK8 was used to detect the inhibitory effect of CXCL1 on tumor proliferation induced by TM in hepatocellular carcinoma .Results pEGFP-C1-CXCL1 was vertified by sequencing analysis .Fluorescence microscopy showed that pEGFP-C1-CXCL1 was transfected into 293T.CXCL1 expression was detected by Western blotting .CCK8 showed that TM inhibited tumor proliferation , while overexpression of CXCL1 decreased the inhabitory rate on cell proliferation of HepG 2 cells under ER stress compared to pEGFP-C1 group and the control group .Conclusion A recombinant pEGFP-C1-CXCL1 plasmid is successfully constructed that can be expressed stably in human 293T cells.Overexpression of CXCL1 can effectively reduce the inhabitory rate of HCC cells induced by the ER stress.
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Activation of microglia plays a vital role in the initiation and maintenance of specific neuropathic pain states. By activating microglia in central nervous system, Toll-like receptor 4 (TLR4) can promote the release of proinflammatory cytokines and neuroactive compounds, participate in the initiation and maintenance of neuropathic pain, and trigger the opiate side effects. Therefore, TLR4 may be a potential therapeutic target for neuropathic pain. Inhibition of TLR4 has shown some biological effects in neuropathic pain models and ibudilast (the TLR4 pathway-inhibiting agent) has been approved for for phase 2 clinical trials. This article briefly reviews the structure, function, and mechanism of TLR4 as well as the development of TLR4-targeted drugs.
Subject(s)
Humans , Neuralgia , Drug Therapy , Toll-Like Receptor 4 , PhysiologyABSTRACT
Objective To observe the effect of intrathecal injection of siRNA targeting Toll-like receptor 4 (TLR4) on neuropathic pain and spinal cord levels of TLR4, IL-1β, and TNF-α in rat model of chronic constriction injury (CCI). Methods Male Sprague-Dawley rats were randomly divided into four groups (n = 10): the sham group (intrathecal normal saline, IT NS), CCI group (CCI+IT NS), mismatch siRNA group (CCI+IT mismatch siRNA), and siRNA-TLR4 group (CCI+IT siRNA-TLR4). The lumbar intrathecal catheters were implanted in rats and CCI models were established as previously described. The TLR4 siRNA were administered intrathecally for 7 days starting from 1 day before surgery. The spinal cord expression of TLR4 mRNA was assessed by real-time PCR. Levels of TNF-α and IL-1β in spinal cord were detected by ELISA. The thermal and mechanical nociceptive thresholds were assessed by paw withdrawal latency (PWL) to radiant heat and von Frey filaments. Results Compared with the sham group, animals in CCI group had significantly lower mechanical and thermal pain thresholds, higher expression of TLR4 mRNA and levels of IL-1β, TNF-α in the spinal cord (P<0.05). Rats in the siRNA-TLR4 group had significantly higher mechanical and thermal pain thresholds (at 1, 3, and 7 days after ligation, P<0.05) and significantly lower expression of TLR4 mRNA and levels of IL-1β, TNF-α in the spinal cord compared with those in the CCI group and mismatch siRNA group(P<0.05). Conclusion Intrathecal injection of siRNA-TLR4 can decrease the levels of inflammatory factors by silencing the TLR4 in the spinal cord of rats, and subsequently relieve the neuropathic pain induced by CCI.