Effect And Mechanism Of Yuan-Zhi-San (YZS) On Degradation Of Hyperphosphorylated Tau Protein In Alzheimer’s Disease

Alzheimer’s disease(often shortened to “AD”)is the most common neurodegenerative disease in the aging population with high morbidity,high mortality,low remission rate,and high medical burden.Currently,there is no effective diseasemodifying therapy for AD.The accumulation of hyperphosphorylated tau protein to form neurofibrillary tangles(NFTs)is a typical pathological feature of AD.How to promote the degradation of hyperphosphorylated tau protein and inhibit its aggregation to form NFTs has been a hot research topic.Ubiquitin-proteasome system(UPS)is a crucial mechanism for the degradation of abnormal or toxic protein in neurons.Hence,UPS pathway with 26 s proteasome as the core promotes the degradation of phosphorylated tau protein.Firstly,with Aβ1-40-induced AD rats as the carrier,the effectiveness of Yuan-Zhi-San(YZS)was observed by detecting the learning and memory ability,brain histomorphology and brain ultrastructure of AD models.Based on the theory of the ubiquitin-proteasome system(UPS)regulates the degradation of hyperphosphorylated tau protein,we proposed a hypothesis that YZS regulates the degradation of hyperphosphorylated tau protein through UPS of AD rats.Secondly,with SAMP8 mouse as the carrier,the effectiveness of YZS on hyperphosphorylated tau protein was observed after the UPS in AD models was inhibited.This study was designed to explore the effectiveness and underlying mechanism of YZS on AD.In this study,we observed the effectiveness of YZS on the degradation of hyperphosphorylated tau protein in AD model,and further explored its possible mechanism.So as to provide experimental evidence support for the clinical application of YZS.ObjectiveThis study was designed to investigate the effectiveness of Yuan-Zhi-San on AD,and further clarify the underlying mechanism.MethodsThe AD rat model was established by Aβ1-40 injections.After 8 weeks of intervention with YZS,the abilities of rats in learning and memory were examined by Morris Water Maze.The brain tissue,dendrite structure and ultrastructure of rats were respectively observed by HE staining,Golgi-Cox staining and transmission electron microscope.The levels of phosphorylated Tau protein in Ser199,Thr231,Ser396 sites of the hippocampus in rats were examined with IHC and Wb.Expressions of UPS key enzymes such as E1,E2 a,CHIP,UCH-L1 and 26 s proteasome were examined by Wb.The safety of lactacystin were investigated in C57BL/6J mouse.The efficacy of lactacystin on inhibiting 26 s proteasome were observed with PC12 cells.Lactacystin were injected into the hippocampus of SAMP8 mice to inhibit the activity of 26 s proteasome.After 8 weeks of intervention with YZS,the mouse of memory and learning performances were examined using Morris Water Maze.The levels of 26 s proteasome and phosphorylated tau protein in Ser199,Thr231,Ser396 sites in the hippocampus of rats were examined with Wb.The combination of tau protein and tubulin was observed with Tau5/α-tublin IF,and the aggregation of the phosphorylated Tau protein were observed with Thioflavin-S/AT8 IF.Results1.(1)On day 4,compared with SO rats,AD rats demonstrated significantly longer escape latency in the orientation navigation experiment(P<0.01).Compared with those of AD rats YZS-treated rats had shorter escape latency in the orientation navigation experiment(p < 0.05).(2)In the probe test,AD rats demonstrated less crossing times(P<0.01),decreased time in the target quadrant(P<0.01)and shorter swimming routes(P<0.01)compared with SO rats.Compared with those of AD rats YZS-treated rats had greater crossing times,increased time in the target quadrant and lengthier swimming routes(P<0.05,P<0.01).2.H&E staining indicated that,in hippocampus CA1 regions of SO rats,the pyramidal cells layer were structured clearly and arranged tightly,cell membrane and nucleolus were obvious and complete.Compared with SO rats,AD rats shown the less pyramidal cells,deeper nucleus,more vacuole,increased microglial cells,more space between intercellular and the blood vessels.After YZS treatment,the AD rats in pathological changes of brains were significantly rescued.These results suggested that YZS could protect against neuronal loss in AD rats.3.To further examine the neuroprotective effect of YZS against Aβ-induced AD,the density of dendritic spines was measured using Golgi-Cox staining.In the brains of SO rats,the dendrites of the cortex cells and hippocampus pyramidal cells were longer with more branches and dense arranged compared with AD rats.Compared with the SO rats,the number of dendritic arborization in AD rats was significantly reduced with the looser arrangement.Compared with those of SO rats,the density of dendritic spines in AD rats decreased significantly(P< 0.01).Compared with AD rats,the density of dendritic spines in rats of M-YZS and H-YZS groups were increased(P < 0.01,P < 0.05).Compared with AD rats,the density of cortical spines in rats with L-YZS treatment was increased(P < 0.05),but the density of dendritic spines in the hippocampus had no significant change(P > 0.05).4.The ultrastructural examination of nerve cells in the hippocampus were examined using the TEM.In the hippocampus CA1 area of SO rats,the nucleus were large and round,the nucleolus were obvious,the euchromatin were rich,and the arrangement of nuclear membrane bilayer membrane were clear and continuous.There were abundant ribosomes in cytoplasm,smooth mitochondrial membrane,cristae or tubules,and no swelling of endoplasmic reticulum in SO rats.In the brains of AD rats,the nuclear morphology were irregular,the nuclear membrane were invaginated with chromatin gathered on the nuclear membrane.Besides,part of the cytoplasm were dissolved with open space,organelle damage,membrane discontinuity and local rupture.Compared with AD rats,the ultrastructural damage of YZS-treated rats were rescued.5.Effectiveness of YZS on hyperphosphorylated tau protein in hippocampus of AD Rats.There was no significant difference in the expression levels of tau5 protein in the hippocampus of rats examined using IHC and Wb(p>0.05).Compared with SO rats,the expression levels of p S199,p S396,and p T231 proteins in the hippocampus of AD rats were significantly increased(p<0.001),which indicated that the phosphorylated degree of tau protein in the hippocampus of AD rats was significantly increased.The results of IHC shown that,compared with the AD rats,the expression levels of p S199 and p T231 protein in the YZS treated rats were reduced(p<0.05,p<0.01),which still significantly higher than the SO group(p<0.01,p<0.001).Compared with AD rats,except for 231 protein of L-YZS-treated rats,the expression levels of p S199 and p T231 of YZS treated rats were decreased(P < 0.05,P < 0.01),which were still significantly higher than SO rats(P < 0.05,P < 0.01).The expressions of p S396 protein of H-YZS-treated rats were lower than AD rats(P <0.05).There was no significant difference between M-YZS-treated rats and L-YZStreated rats.6.The effectiveness of YZS on the expression of key enzymes of UPS in the hippocampus of AD rats.Compared with the SO rats,the expression levels of Ub E1a/b,Ub E2 a,CHIP,UCH-L1,26 s proteasome were significantly reduced in AD rats(p<0.05,p<0.01,p< 0.001),which indicated the dysfunction of UPS.Compared with the AD group,the expression levels of Ub E1a/b,Ub E2 a,CHIP,26 s proteasome,and UCH-L1 protein in YZS-treated rats increased(p<0.05,p<0.01,p<0.001),which indicated that YZS can rescue the damage of UPS dysfunction in the hippocampus of AD rats.The YZS can rescue reduce the phosphorylation levels of tau protein by regulating the UPS pathway.7.Effectiveness of YZS on learning and memory ability of SAMP8 mice after inhibiting 26 S proteasome.Compared with SAMR1 mice,the average escape latency of AD rats were significantly prolonged(p<0.001).After 8 weeks treatment of YZS,the escape latency of mice was shortened(p<0.05,p<0.01).Compared with AD mice,the escape latency of LAC mice were further prolonged(p<0.05).Compared with LAC mice,the escape latency of LAC+YZS rats was shortened(p<0.05).However,there were no significant difference between LAC+YZS mice and AD mice(p>0.05),and there were no significant difference between LAC+YZS mice and AD mice(p>0.05).The results indicated that,even though the 26 S proteasome of SAMP8 mice was inhibited,YZS could still improve the learning and memory ability of SAMP8 mice.And these effectiveness of YZS were partially inhibited.8.The effectiveness of YZS on the pathological changes of phosphorylated tau protein in SAMP8 mice after the intervention of lactacystin.Wb results indicated that there was no significant difference about the expression level of Tau5 protein in hippocampus of mice in each group(p>0.05).Compared with SAMR1 mice,the protein expression levels of p S199,p T231,and p S396 in AD mice increased significantly(p<0.001,p<0.01).Compared with these AD mice,the expression levels of p S199,p T231,and p S396 protein of YZS-treated mice were reduced(p<0.05,p<0.01).Compared with the AD mice,the expression levels of p S199 and p T231 proteins of LAC mice were significantly increased(p<0.05).Compared with the AD mice,the expression levels of p S396 proteins in LAC mice were no significant difference(p>0.05).Compared with the LAC mice,the protein expression levels of p S199,p T231,and p S396 protein in the LAC+YZS mice were decreased(p<0.05,p<0.01).Compared with the AD mice,the expression of p S396 protein were still decreased in LAC+YZS mice(p>0.05).Compared with the AD mice,the expression of p S199 and p T231 proteins in LAC+YZS mice were no significant difference.The Tau5/α-tublin IF indicated that the colocalization fluorescence intensity of AD mice was significantly weaker than that of SAMR1 mice.Compared with AD mice,the intensity of YZS-treated mice significantly increased.Compared with the AD mice,the intensity of the LAC mice further decreased.Compared with the LAC mice and the AD mice,the intensity of the LAC+YZS mice increased.Compared with the LAC mice,LAC+YZS mice were still significantly lower.The results of Thioflavine-S/AT8 IF staining indicated that,compared with SAMR1 mice,the fluorescence intensity of Thioflavin-S and AT8 in AD mice significantly increased,and the colocalization fluorescence coloration also significantly increased.Compared with AD mice,the fluorescence intensity of Thioflavin-S,AT8 and the colocalization intensity in YZS-treated mice significantly decreased.Compared with AD mice,the single fluorescence coloration area and intensity of Thioflavin-S/AT8 colocalization in AD mice were significantly increased.Compared with LAC group,the fluorescence intensity in LAC+YZS mice decreased.Compared with YZS mice,the fluorescence intensity in LAC+YZS mice still significantly enhanced.The results indicated that after the 26 S proteasome of SAMP8 mice was inhibited,YZS could partly,rather than completely,rescue the hyperphosphorylation of tau protein,the binding between tau protein and tubulin,and the abnormal aggregation of tau protein in SAMP8 mice.Conclusion1.YZS rescues the memory deficit of AD models,ameliorates the histopathology and ultrastructural abnormalities,and enhances the density of dendritic spines in AD models.2.YZS improves the degradation of hyperphosphorylation of tau protein and modulates the expression levels of UPS-related proteins in AD rats,which mean Yuanzhisan may modulates UPS to promote the degradation of phosphorylated tau protein and inhibit the aggregation.3.UPS is the key but not the single target of YZS to rescues the pathological changes of tau protein and then improve the learning and memory ability of AD models.