OGD Induced DsRNA Activating NLRP3 Signaling Pathway And Its Related Inflammatory Factor In The Role Of Brain Damage Mechanism

The prime pathological mechanism of cerebral ischemic injury is neuronal damage, which is one of the key causes of serious disability. The causes of neuronal injury are not only induced by exogenous stimulus resulting in the dysregulation of protein expression and signaling pathways, but also by the change of endogenous transcript expression. Recently, research reported that double-stranded RNA(ds RNA) may be a dangerous signal, through which could trigger cellular reaction and finally lead to apoptosis. However, whether ds RNA plays as an endogenous dangerous signal in neuronal injury is still unknown. And the mechanisms of ds RNA are not clear, which starve for investigation. Therefore, our objectives are investigating the effects and mechanisms of ds RNA in cerebral ischemic injury.PartⅠ OGD induced HT22 cells ds RNA in the form of B1 RNAObjectives: To verify the endogenous ds RNA after OGD and identify the components and sequence of ds RNA in HT22 cells. Methods: 1.We established OGD model in HT22 cells and neuronal ischemic model in vitro. We observed neurons under light microscope in normal condition and after OGD treatment individually. Then we detected LDH to evaluate the model. 2. ds RNA expression was labeled by immunofluorescence. After ds RNA was labeled with J2 antibody and nucleus was stained with DAPI, the merged pictures were observed by fluorescence microscope. 3. Comparing with B1 RNA sequence of mice in established ds RNA of c DNA library. Sequencing analysis was using IP, q RT-PCR, RT-PCR, T-A clone, and agarose gel electrophoresis experiment. Results: 1. 6 h after OGD, HT22 was observed under light microscope. It was observed that changed nucleus membrane, shrunk cell, nuclear shrinkage, swelling r ER and mitochondrial. Cell injury was increased. Compared to control, LDH level was dramatically increased in OGD group. The OGD model was built successfully. 2. Compared to control, the ds RNA connected with J2 antibody, which mainly distributed in cytoplasm around nucleus, was increased in OGD 24 h group under fluorescence microscope. 3. Results of sequencing analysis showed that ds RNA produced mainly 215 bp B1 RNA after OGD, which sequence was in accordance with B1 RNA and B1 DNA of mice. Results of agarose gel electrophoresis experiment and q RT-PCR showed that ds RNA was mainly composed of B1 RNA. Conclusion: ds RNA was produced in HT22 cells after OGD, which was mainly B1 RNA and the sequence was in accordance with B1 of mice.PartⅡ B1 RNA and NLRP3 of the expression in OGD and MCAO modelObjectives:To verify B1 RNA expression and NLRP3 inflammation pathway in mice primary cortex neuronal OGD model in vitro and middle cerebral artery occlusion(MCAO) model in vivo. Methods: 1. Establishing primary neuronal OGD model and MCAO model in mice. After OGD, we observed neuronal morphology, detected LDHlevel, scored by neurological function and stained with TTC. 2. Mice neuronal B1 RNA and NLRP3 expression were detected by q RT-PCR. NLRP3 expression was detected by western blot. In cortex primary neuron of pc DNA3.1-B1 transfected mice, B1 RNA transcription and NLRP3 expression were detected through MTS experiment, q RT-PCR and western blot. Results: 1. Compared to normal primary neurons, at 2 h after OGD we observed neuronal injury induced the shortening or disappearing of axon and the dramatically increased LDH level. Neurological function score showed there were neurological abnormalities in MCAO mice. And TTC staining showed the pale white zone in MCAO mice, which indicated the model was established successfully. 2. Fluorescence showed ds RNA and NLRP3 were overexpressed significantly in neurons of MCAO mice. Results of q RT-PCR and western blot showed B1 and NLRP3 expression were increased in MCAO mice.In OGD and pc DNA3.1-B1 group, NLRP3 protein expression was significantly higher than control. However, in pc DNA3.1-B1+si NLRP3 group, NLRP3 protein expression was significantly lower than pc DNA3.1-B1 group. Meanwhile, in OGD+si NLRP3 group, NLRP3 protein expression was lower than OGD group. This result indicated that we successfully interfered NLRP3 expression which was induced by OGD and B1 RNA in our experiment. MTS experiment showed OGD and transfected pc DNA3.1-B1 could significantly inhibit primary neuron growth. Interfering of NLRP3 expression in OGD and pc DNA3.1-B1 group could improve cellular viability. Conclusions: In OGD and MCAO model, B1 RNA and NLRP3 expression were upregulated in primary cortex neurons. Increased B1 RNA and NLRP3 could inhibit mice primary cortex neuron growth.PartⅢ Expressions and connections between B1 RNA and NLRP3 pathway-regulated inflammation factors in HT22 OGD model.Objectives: The expression of B1 and NLRP3 regulated inflammatory factors in ischemic cerebral injury. Methods: After transfecting pc DNA3.1-B1 and pc DNA3.1-B1+si NLRP3, we used Western blot,q RT-PCR,ELISA to detect the expression of NLRP3, IL-1β, and TNF-α in OGD-treated HT22 cells. MTS and ELISA were performed to detect cellular viability and the expression of pro-inflammatory factors after transfecting pc DNA3.1-B1 and pc DNA3.1-B1+si NLRP3 plasmid. Results: The results of western blot showed that the expression of NLRP3 was increased depending on OGD time. Results of q RT-PCR showed NLRP3 pathway was activated after OGD in HT22 cells. And the results of ELISA indicated that the expression of IL-1β and TNF-α were increased at 12 h after OGD. NLRP3 was increased in HT22 cells after transfected with B1. Compared to control and blank vector-transfected groups, the expression of NLRP3, IL-1β, and TNF-α was higher in pc DNA3.1-B1 transfected group. Results of ELISA indicated that pc DNA3.1-B1 transfected cells had a higher expression of IL-1β, and TNF-α than control and blank vector-transfected groups. Results of MTS showed that OGD group had a lower cellular viability than control group. pc DNA3.1-B1 transfected group had a lower cellular viability than control and blank vector-transfected groups. However, OGD+ si NLRP3 group had a higher cellular viability than OGD group. pc DNA3.1-B1+si NLRP3 group had a higher cellular viability than pc DNA3.1-B1 transfected group. Conclusions: B1 RNA was participated in activating NLRP3-upregulated IL-1β and TNF-α after OGD in HT22 cells, therefore, promoted the process of cell injury.