Study On The Anti-apoptotic And Anti-inflammatory Activities And Mechanisms Of Diterpene Acids

Alzheimer’s disease（AD）,also known as Senile dementia,is a common central nervous system degenerative disease of the elderly.How to prevent AD has been one of the world’s difficult problems.In the previous study of our group,it was found that the diterpene acids from the root bark of Pseudolarix kaempferi Gorden,pseudolaric acid A（PAA）and pseudolaric acid B（PAB）could alleviate insulin resistance,endoplasmic reticulum stress and mitochondrial damage;at the same time,it could reduce the NO release of LPS induced mouse macrophages（RAW264.7）and mouse microglia（BV2）significantly,and protect the apoptosis ofβcell induced by palmitic acid.In this study,we further studied the protective effect of PAA and PAB on the apoptosis of rat nerve cells（PC12）induced by Aβ_25-35,the anti-inflammatory reaction of macrophages and microglia,and the mechanism of the above effects.Firstly,the apoptosis model of PC12 induced by Aβ_25-35（30μM）was established.Then,the effects of PAA and PAB（0.001-10μM）on PC12 cells viability and the inhibition of cells apoptosis were researched.MTT analysis was used to research the effect of PAA and PAB on the viability of s PC12 cells harmed by Aβ_25-35,AO/EB and Annexin V-FITC/PI fluorescent staining was used to research the inhibiting effect of PAA and PAB on apoptosis of PC12 cells and western blot was used to research the anti-apoptosis mechanism of PAA and PAB for nerve cells.The results showed that,compared with Aβ_25-35 model group,the survival rate of cells treated with PAA or PAB together with Aβ_25-35 increased significantly,and the apoptosis of cells decreased significantly.Meanwhile,the results of western blot showed that PAA and PAB could up-regulate the expression of IRS-1,P-Akt/Akt,and down regulate the expression of P-GSK-3β/GSK-3β.These suggested that PAA and PAB could inhibit the apoptosis of PC12 cells induced by Aβ_25-35 by regulating the expression of IRS-1,P-Akt and P-GSK-3β.The inflammatory model was established by LPS-stimulated mouse BV2microglia and RAW264.7 macrophages.Cells were treated with different concentrations of PAA and PAB,and then the effects of PAA and PAB on inflammation were investigated.Cell migration was investigated by the wound healing assay.NO released from the cells was tested by Griess assay.ROS level in the cells was tested by fluorescence spectrophotometer.mi RNA-155 expression by in the cells was detected by real-time PCR.Protein expression of P-Iκ-Bα/Iκ-Bα,TNF-α,and TGF-β1 was detected by by Western blot.The results showed that the cell migration,NO release level,ROS level,and mi RNA-155 expression in the cells of PAA or PAB treated group were significantly lower than those in the cells of LPS-stimulated group.Furthermore,the results of western blot showed that PAA or PAB could down regulate the expression of P-Iκ-Bα/Iκ-Bα,TNF-αand TGF-β1.These suggested that PAA and PAB could alleviate the inflammatory response of macrophages and microglia induced by LPS.In conclusion,both of PAA and PAB can inhibit the apoptosis of PC12 cells induced by Aβby up-regulating the insulin receptor-PI3K/Akt signaling pathway.Meanwhile,both of them can also inhibit the inflammatory reaction of mouse macrophages and microglia,which may alleviate the injury of neurons caused by inflammation.PAA and PAB have the potential to prevent and treat Alzheimer’s disease,and have the value of further research.