Anti-inflammatory Effect Of Puerarin On Focal Cerebral Ischemia/reperfusion Injury Via Down-regulating TLR4Mediated NF-κB Signaling Pathway

Objective：Emerging evidence has demonstrated that innate immune and inflammatoryresponses play a critical role in focal cerebral ischemia/reperfusion (I/R) induced neuronal damage.Toll-like receptor-4(TLR4), nuclear factor kappa-B (NF-κB) and tumor necrosis factor (TNF-α)have been linked to inflammatory reactions. It has been reported puerarin may represent a novelapproach to functional protection in cerebral I/R injury related disorders. Until now themechanisms underlying the neuroprotective effects of puerarin on inflammatory response inducedby cerebral I/R injury have not yet been understood. So, in our study, we intend to confirm thatpuerarin exerted anti-inflammatory effect on ischemia/reperfusion brain via down-regulating TLR4medicated NF-κB signaling pathway.Methods:1. Male SD rats (250g-280g) were randomly divided into sham operation group, focalcerebral I/R (vehicle) group and puerarin treatment group.90min cerebral ishemia/reperfusion wasinduced by middle cerebral artery occlusion (MCAO) model with an endovascular filament;2. Inthe puerarin treatment group, different doses of puerarin (50mg/kg,100mg/kg,200mg/kg) wereadministered intraperitoneally at30min before MCAO and8h after reperfusion. The rats in thevehicle group and sham operation group received same dose of saline replacing puerarin with samemethod at the same time;3. At24h after MCAO, neuronal damage, infarct size and brain watercontent were measured;4. At24h after MCAO, the expression of TLR4, MyD88, NF-κB andTNF-α in different groups was analyzed by RT-PCR and immunohistochemistry method.Results:1.Puerarin attenuated the neurological defect score (NDS), infarct size and water content of ischemic brain tissue after MACO:1-1.The neurological defect score (NDS) at24hours after MCAO significantly reduced inpuerarin (100mg/kg) treatment group VS. vehicle group(p=0.023). The NDS decreased in the50mg/kg treated and200mg/kg treated groups compared with the vehicle group also, but failed toreach the statistical differences.1-2. Infarct size was observed at24hours after MCAO using vital staining with2%2,3,5-triphenyltetrazolium chloride (TTC). No infarction was observed in Sham group, while extensiveinfarction developed in both striatum and cortex after MCAO. The relative infarct size, thepercentage of infarct region occupying the whole brain tissue area, decreased in the puerarin(100mg/kg) treatment group compared with the vehicle group at24hours(p=0.014).1-3. Brain Water content at24hours after MCAO was measured by wet–dry method. Watercontent in the puerarin (100mg/kg) treatment group decreased significantly at24hours comparedwith that in the vehicle group(p=0.026).2.Puerarin reduced the expression of TLR4, MyD88, NF-κB and TNF-α mRNA after MACO:The expression of TLR4, MyD88, NF-κB and TNF-α mRNA in the brain tissue of each groupwas detected by RT-PCR at24hours after MCAO. The level of TLR4mRNA expression washigher in the vehicle group than that in the sham group(p=0.007), and was lower than that in thepuerarin treatment group(p=0.025). The expression of MyD88, NF-κB and TNF-α mRNA24hours after MCAO was higher in the vehicle group than that in the sham group and the puerarintreatment group(Myd88: p=0.048; NF-κB p=0.04; TNF-α: p=0.029).3. Puerarin reduced the expression of TLR4, MyD88, NF-κB and TNF-α protein after MACO:The expression of TLR4, MyD88, NF-κB and TNF-α protein in the brain tissue was detectedby an immunohistochemical technique24hours after the MACO. Few cells were stained withTLR4in the cortex and striatum in sham group. In vehicle group, the number of positive cells ofTLR4significantly increased in the ischemic cortex and striatum. In100mg/kg puerarin treatmentgroup, the number of positive cells of TLR4was significantly decreased compared with vehiclegroup(cortex：p<0.05；striatum：p<0.05). The number of positive cells of MyD88, NF-κB andTNF-α was significantly increased in the vehicle group than that in the sham and puerarintreatment group(cortex Myd88: P<0.05; NF-κB: P<0.05; TNF-α:P<0.05striatum Myd88:P<0.05; NF-κB: P<0.05; TNF-α: P<0.05). Conclusions: our study provided new mechanism into the protective effect of puerarin on focalfocal cerebral ischemia/reperfusion Injury. This effect was associated with the depressedexpression of TLR4, MyD88, NF-κB and TNF-α in the acute phase of focal cerebralischemia/reperfusion injury. Anti-inflammatory via down-regulation of TLR4-MyD88-NF-κBactivation pathway might be a potential mechanism of puerarin’s neuroprotection onischemia/reperfusion injury.