Microglial inflammation, involved in the occurrence and development of sepsis-associated encephalopathy, exhibits upregulation of proinflammatory cytokine and proinflammatory enzyme expression, leading to inflammation-induced neuronal cell apoptosis. TIR domain containing adaptor molecule-2 (TICAM-2) participates in lipopolysaccharide (LPS) mediated BV2 cell inflammation. SET8 plays a crucial role in a variety of cellular signal pathways. In this study, we hypothesize that SET8 participates in LPS-mediated microglial inflammation via modulation of TICAM-2 expression. Our data indicated that LPS induced BV2 inflammation via upregulation of TICAM-2 expression. Moreover, LPS treatment inhibited SET8 expression, while it increased activating transcription factor 2 (ATF2) expression. The effects of sh-SET8 and ATF2 overexpression were similar to that of LPS treatments. Inhibition of TICAM-2 expression counteracted sh-SET8-mediated and ATF2 overexpression mediated BV2 cell inflammation. Further, SET8 was found to interact with ATF2. A mechanistic study found that H4K20me1, a downstream target of SET8, and ATF2 enriched at the TICAM-2 promoter region. Luciferase reporter assays indicated that sh-SET8 increased TICAM-2 promoter activity but augmented the effect of ATF2 overexpression on TICAM-2 promoter activity as well. Co-transfection of sh-SET8 with ATF2 overexpression more dramatically increased TICAM-2 expression in BV2 cells. The present study indicated that SET8 interacted with ATF2 to modulate TICAM-2 expression, which participated in LPS-mediated BV2 cell inflammation.
Objective To observe the analgesic effects of lentivirus-mediated RNA interference of the expression of glial cell line-derived neurotrophic factor (GDNF) on bone cancer pain.Methods (1)Lvs-siGDNF was constructed and bone cancer pain models were established by intra-tibial injection of MRMT-1 cells.(2) The rats were randomly divided into 4 groups according to different drugs which were infused into subarachnoid space through the intrathecal tubes:psiHIV-U6,psiHIV-GDNF-mU6,mor-phine,and normal saline.(3) The pain threshold at different time points (normal rats,7 days and 14 days after dosing) was measured.(4) The changes of pain threshold were observed from preoperative day to 7 days and 14 days after administration.The levels of GDNF,glial fibrillary acidic protein (GFAP),and Fos were detected by immunofluorescent staining technique and Western blotting.Results (1) As compared with control group (0.117 ±0.060) and transfection group (0.113 ±0.072),GDNF siRNA sequence 3 (0.045 ± 0.034) was the most effective interference segment confirmed by Q-PCR (P ＜ 0.05).The percentage of fluorescent cells to the brightfield was 0.87.TLvs-siGDNF was 3.48 × 108 pfu/ml.(2)Thermal pain threshold at 7th day (3.80 ±0.69) and 14th day (3.71 ± 1.10),and mechanical pain thresh-old at 14th day (2.58 ± 1.93) were all increased in siGDNF group than normal saline group (1.38 ± 1.01,1.11 ± 1.02,and 1.53 ±0.69) and psiHIV-U6 group (1.39 ± 1.18,0.67 ±0.27,and 1.21 ± 1.06)(P ＜ 0.05),while there was no significant difference in mechanical pain threshold at 7 day after administration (P ＞ 0.05).(3) RNA interference effectively decreased the expression level of GDNF in siGDNF group (P ＜0.05).(4) As compared with other groups,the expression levels of Fos and GFAP in the spinal dorsal horn were decreased in morphine group and siGDNF group (P ＜ 0.05).Conclusion The analgesic effect of siGDNF is similar to that of morphine in bone cancer rats,and siGDNF worked effectively more than 2 weeks.SiGDNF realized analgesic effect probably by inhibiting the astrocyte activation.
Key words:
Bone cancer pain; Glial cell line-derived neurotrophic factor; RNA interference; Lentiviral
To construct recombinant over expression vector of Homo sapiens proenkephalin (PENK) gene and explore the function of PENK gene.Fragment containing PENK ORF gene was inserted into vector plasmid HIV, then the recombinant over was confirmed by enzyme digestion and sequencing. Lentivirus containing the recombinant over expression vector was produced by virus packaging with 293Ta cell,and then the lentivirus was transfected into HT1080 cell and the virus titer was estimated. The PC12 were tansfected with resulting lentivirus and un-transfected PC12 cells as control. The images of the PC12 cells were captured at 48 h post-transfection and the number of cells was also evaluated; the changes of PENK mRNA in transfection and control group were measured with RT-PCR.The constructed PENK ORF recombinant over expression vector was confirmed by enzyme digestion and sequencing. The number of PC12 cells in transfection and control group at 48 h post-transfection was 127.93 +/- 2.48 and 88. 60 +/- 2.55 respectively, and the statistical difference between them was observed (P < 0.01).Recombinant over expression vector of PENK gene was successfully constructed and the PENK gene can promote the growth of PC12.
Bone cancer pain is a common symptom in cancer patients with bone metastases and the underlying mechanisms are largely unknown. The aim of this study is to explore the endogenous analgesic mechanisms to develop new therapeutic strategies for bone-cancer induced pain (BCIP) as a result of metastases. MRMT-1 tumor cells were injected into bilateral tibia of rats and X-rays showed that the area suffered from bone destruction, accompanied by an increase in osteoclast numbers. In addition, rats with bone cancer showed apparent mechanical and thermal hyperalgesia at day 28 after intratibial MRMT-1 inoculation. However, intrathecal injection of morphine or lentivirus-mediated glial cell line-derived neurotrophic factor RNAi (Lvs-siGDNF) significantly attenuated mechanical and thermal hyperalgesia, as shown by increases in paw withdrawal thresholds and tail-flick latencies, respectively. Furthermore, Lvs-siGDNF interference not only substantially downregulated GDNF protein levels, but also reduced substance P immunoreactivity and downregulated the ratio of pERK/ERK, where its activation is crucial for pain signaling, in the spinal dorsal horn of this model of bone-cancer induced pain. In this study, Lvs-siGDNF gene therapy appeared to be a beneficial method for the treatment of bone cancer pain. As the effect of Lvs-siGDNF to relieve pain was similar to morphine, but it is not a narcotic, the use of GDNF RNA interference may be considered as a new therapeutic strategy for the treatment of bone cancer pain in the future.
Hyperglycemia mediates oxidative stress, thus inducing transcription factor nuclear factor kappa B (NF-κB) activation, increasing endothelial adhesion molecule expression and monocyte/endothelial interaction, and resulting in endothelial injury. Ketamine was reported to attenuate oxidative stress in many cases. In this research, we determined whether and how ketamine protects against high-glucose-mediated augmentation of monocyte/endothelial interaction and endothelial adhesion molecule expression in human umbilical vein endothelial cells. High glucose augmented monocyte/endothelial adhesion and endothelial adhesion molecule expression. High glucose induced reactive oxygen species (ROS) production and augmented phospho-protein kinase C (p-PKC) βII expression and PKC activity. Moreover, high glucose inhibited the inhibitory subunit of nuclear factor-κBα (IκBα) expression in the cytoplasm and induced NF-κB nuclear translocation. Importantly, the effects induced by high glucose were counteracted by ketamine treatment. Further, CGP53353, a PKC βII inhibitor, inhibited high-glucose-mediated NF-κB nuclear translocation, attenuated adhesion molecule expression, and reduced monocyte/endothelial interaction. Further, these effects of ketamine against high-glucose-induced endothelial injury were inhibited by phorbol 12-myristate 13-acetate, a PKC βII activator. In conclusion, ketamine, via reducing ROS accumulation, inhibited PKC βII Ser660 phosphorylation and PKC and NF-κB activation and reduced high-glucose-induced expression of endothelial adhesion molecules and monocyte/endothelial interaction.
Hyperglycemia-induced endothelial inflammation participates in the pathogenesis of cardiovascular complications in diabetics. Previous studies showed that protein tyrosine phosphatase 1B (PTP1B) and ETS proto-oncogene 1 (ets1) are involved in hyperglycemia-induced endothelial inflammation. In this study, we hypothesized that ets1 modulates PTP1B expression, thus playing a crucial role in hyperglycemia-induced vascular endothelial inflammation. Our results indicated that high glucose increases monocyte/endothelial adhesion, vascular cell adhesion molecule-1 (VCAM-1) expression and p65 phosphorylation in human umbilical vein endothelial cells (HUVECs). Moreover, high glucose-mediated endothelial inflammation is reversed by PTP1B silencing. In addition, high glucose increases ets1 expression in HUVECs. silencing reverses high glucose-mediated endothelial inflammation. Furthermore, the effect of ets1 overexpression is similar to that of high glucose treatment, which is counteracted by si-PTP1B. The results from ChIP assays indicated that ets1 occupies the PTP1B promoter region. Ets1 overexpression enhances PTP1B promoter activity, which is disappeared after specific binding site mutation. experiments demonstrated that the expressions of VCAM-1, PTP1B, and ets1, as well as the phosphorylation of p65 are augmented in the aorta of diabetic rats. In conclusion, ets1 contributes to hyperglycemia-mediated endothelial inflammation via upregulation of PTP1B expression.
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