To Analyze The Molecular Mechanism Of Anti-inflammatory Of Panax Notoginseng Saponins In Cerebral Ischemia From The Perspective Of Regulating RIG-I Signaling Pathway

Objective:To observe the effects of total saponins ofpanax notoginseseng on RIG-I signaling pathway related molecules RIG-I?Traf2 and NF kappa B expression in ischemic brain injury as well as the moderating role of downstream inflammatory factor TNF-? and IL-8, in revealed total saponins of panax notoginseng in cerebral ischemia play the molecular mechanism of the anti-inflammatory effect, at the same time also for RIG-I signaling pathway in ischemic brain injury and provide further experimental evidence.Mothods:1. Using established in our laboratory rat brain microvascular endothelial cells (brain microvascular endothelial cells, BMECs) primary culture technique, will be transmitted to the thrid generation of cells and adopt the method of oxygen glucose deprivation (oxygen-glucos-deprivation, OGD) preparation to ischemic injury model.2. To observe the effects of Panax notoginseng saponins intends to ischemic injury in rat brain micro vascular endothelial cells RIG-I signaling pathway effects:cerebral microvascular endothelial cells of rats divided into four groups:normal groups model groups PNS group and RIG-I siRNA group. Except the normal group, the rest of the group by OGD method modeling, PNS group in modeling before and in the process of adding a final concentration of 22ug/ml of total saponins ofpanax notoginseng; RIG-I siRNA group, before modeling using gene transfection technique, import RIG-I siRNA RIG-I expression, modeling after the success of transfection After the modeling, real-time fluorescence quantitative PCR (RT-FQ-PCR) and Western blotting (Western Blot) were used to detect the expression of RIG-I?Traf2 and NF-?B mRNA and protein in the RIG-I signaling pathway in each group.3. Observation of PNS on ischemic injury of rat microvascular endothelial cells RIG-I signaling pathways downstream of inflammation factor expression:The influence of the cell group up with processing, using ELIS A method to detect the inflammatory factor TNF-??IL-8 levels in each group cells.4.To investigate the effects of PNS on lipopolysaccharide (lipopolysaccharide, LPS) activation of RIG-I signaling pathway:To further validate regulate RIG-I signaling pathway is effective interventions target of PNS, we used another RIG-I pathway activation model that LPS stimulation model was regulation of the model of PNS-induced RIG-I pathway in each molecule expression. The three generations of cultured rat brain microvascular endothelial cells were divided into three groups, namely control group?LPS group and LPS+PNS group. In addition to the normal group, the other two groups added to a final concentration of lug/ ml LPS, LPS+PNS group added to a final concentration before and during the activation of PNS 22?g/ml, using RT-FQ-PCR and Western Blot method method to detect the expression RIG-I?Traf2 and NF-?B mRNA and protein expression for each group cells.5.Observe the RIG-I and NF-kappa B expression in animal experiments:Rats divided into sham operation group?model group (permanent middle cerebral artery occlusion model) and PNS group, which PNS group before making model 48h,24h administration, administered 2 h after modeling and in the 24 h sampling; sham operation group and model group at the same time points to medicine material, but is administered saline.The expression of RIG-I and NF-?B in rat cerebral vascular endothelial cells was detected by immunohistochemistry.Results:1. PNS intends to ischemic cerebral microvascular endothelial cells RIG-I signaling pathway is down:After model of oxygen glucose deprivation, RIG-I signaling pathway is activated, the RIG-I?Traf2 and NF kappa B mRNA and protein expression levels were significantly increased; RIG-IsiRNA group the factor expression was significantly down regulated; compared with the model group, total saponins of Panax notoginseng can at mRNA and protein level both effectively reduce OGD induced RIG-I?Traf2 and NF kappa B expression (P< 0.05), shows the similar with RIG-IsiRNA.2. Panax Notoginseng Saponins can reduce quasi ischemic rat brain micro vascular endothelial cell inflammatory factor TNF-a and IL-8 expression:RIG-IsiRNA can effectively reduce OGD brain micro vascular endothelial cells of TNF-a and IL-8 expression, with statistical significance (P< 0.05), suggesting that RIG-I signaling pathway is involved in the inflammatory response of ischemic brain injury. Compared with the model group, the content of PNS group of brain microvascular endothelial cells in TNF-a and IL-8 were significantly reduced (P<0.05)3. Panax Notoginseng Saponins on LPS-induced BMECs RIG-I signaling pathway has a downward effect:After LPS stimulation of endothelial cells, RIG-I?TRAF2 and NF-?B mRNA and protein expression were significantly increased (P<0.05). After the intervention of PNS, the mRNA and protein of RIG-I?TRAF2 and NF-?B expression awere reduced, with significant difference (P<0.05)4. Total saponins of Panax Notoginseng on animal brain artery embolism model after the expression of RIG-I and NF kappa B have a downward effect:After modeling RIG-I and NF kappa B in cerebral micro vessels increased expression was strongly positive; give PNS treatment of RIG-I and NF kappa B in cerebral micro vessels was significantly reduced, weakly positive.Conclusion:1. In the ischemic injury RIG-I signaling pathways activated, then induced downstream inflammation factor TNF-a and IL-8 high expression, prompt the pathways involved in the inflammation process of ischemic brain injury.2. Panax notoginseng saponins by RIG-I signaling pathway in regulating, can reduce the expression of inflammatory cytokines TNF-a and IL-8, suggesting that inhibition after OGD cells RIG-I signaling pathway activation, may total saponins of Panax notoginseng in ischemic brain damage play one of the intrinsic mechanism of the anti-inflammatory effect.3. Panax notoginseng saponins on the activation of LPS induced brain endothelial cells of the RIG-I signaling pathway also has inhibitory effect, further confirmed that RIG-I signaling pathway may be one of the targets to play the anti-inflammatory effect of panax notoginseng saponins.4. In the animal model of middle cerebral artery embolism, panax notoginseng saponin also can reduce the the expression of the RIG-I and NF kappa B in model rats after microvascular, the experimental results are consistent with the cells.