The Improving Effect Of Pentoxifylline On Age-related Neurobehavioral Deficits And Its Mechanism

The proportion of the population that is 60 years old or above is rising globally,and increase in life expectancy accelerates aging of populations.The inevitable and irreversible process of aging is a key element in most neurodegenerative diseases and is characterized by physical deterioration,which impacts both lifespan and quality of life.Most neurodegenerative diseases,such as Parkinson’s disease and Alzheimer’s disease,are characterized by deficits in motor and cognitive functions that are associated with large socioeconomic and personal burdens.As the elderly population increases,the number of individuals living with age-related diseases will grow;effective preventive measures and therapies are therefore urgently needed.Aging is a major factor involved in a gradual decline of brain function.It has been characterized by pathological features such as reactive oxygen species（ROS）-induced oxidative stress,accumulation of oxidative damage molecules（proteins,nucleic acids and lipids）,and mitochondrial dysfunction.Mitochondria are the primary source and target of ROS,which generate ROS as a natural byproduct of oxidative phosphorylation and scavenge ROS by efficient antioxidant defense systems.During aging,endogenous antioxidant capacity is gradually decreased and then overwhelmed by oxidative stress.Moreover,ROS in excess may induce oxidative damage to mitochondrial DNA（mt DNA）and proteins,and cause detrimental effects on mitochondria function,leading to a reduction in mitochondrial quality and content,which potentially accelerate aging and the development of age-associated neurodegenerative diseases.The previous studies have revealed that a decrease in mitochondrial biogenesis during aging would reduce turnover of mitochondrial components,which further weaken and impair mitochondrial function and antioxidant defense systems.Hence,enhancing antioxidative capability,improving mitochondrial dysfunction and promoting mitochondrial biogenesis may act as a rational strategy for maintaining normal brain function of aged organisms.Nuclear factor erythroid 2-related factor 2（Nrf2）is a transcription factor that regulates the expression of antioxidant proteins.It protects cells against oxidative,inflammatory,and metabolic stresses by controlling the expression of a series of detoxification and antioxidant enzymes through antioxidant response element（ARE）.Under normal physiological conditions,Nrf2 is inhibited by cytoplasmic kelch-like ECH-associated protein1（Keap1）and degraded by the proteasome.Under stress conditions,Nrf2 is released from Keap1 and translocates to the nucleus where it binds to ARE,prompting the expression of diverse cytoprotective gene networks to enhance the antioxidant capacity of cells and protect cells from toxic injury,such as heme oxygenase 1（HO-1）and NAD（P）H:quinoneoxidoreductase1（NQO1）.Activation of the Nrf2/ARE pathway protects cells against insults from oxidative stress,and disruption of the Nrf2/ARE pathway leads to increased oxidative damage.Suppression of Nrf2 signaling is a crucial contributor to the premature aging phenotype of Hutchinson-Gilford progeria syndrome,a rare fatal premature aging disorder.In addition,emerging evidence indicates that Nrf2 modulates mitochondrial biogenesis,particularly under stress conditions.Mitochondrial biogenesis is a complex process during which new mitochondria are formed from preexisting mitochondria in the cells to maintain mitochondrial functions and regulate antioxidant defense,and it is primarily regulated by peroxisome proliferator activated receptor-gamma（PPARγ）coactivator 1-alpha（PGC-1α）.In addition to regulating mitochondrial biogenesis by promoting expression of nuclear respiratory factor 1（NRF-1）and mitochondrial transcription factor A（TFAM）,PGC-1αactivates the antioxidant system and increases levels of ROS-detoxifying enzymes under high-ROS conditions.Upregulation of PGC-1αand activation of Nrf2 have therefore been proposed as potential early preventive measures to combat aging and age-related disorders.Pentoxifylline（PTX）is an adenosine antagonist and nonspecific type of4-phosphodiesterase inhibitor.It is a potent antioxidant and modulator of a variety of transmitters and penetrates the blood brain barrier rapidly and efficiently after systemic administration.This drug was initially introduced for the treatment of peripheral circulatory and respiratory disorders,primarily because of its hemorrheologic effects.Recent studies have shown the neuroprotective effect of PTX against a host of neurobehavioral disorders induced by ischemic brain injury and and stroke.Furthermore,as a modulator of intracellular 3’-5’-cyclic adenosine monophosphate（cAMP）signaling pathways,it was found that PTX is able to modulate cellular oxidative stress and mitochondrial biogenesis against nonalcoholic steatohepatitis,major depressive disorder,and cognitive dysfunction.Because oxidative damage and mitochondrial dysfunction are also involved in aging and aging-related neurodegenerative diseases,PTX might serve as a potential intervention to inhibit aging-related processes.In this study,we therefore examined the effects of PTX treatment on antioxidative ability and mitochondrial biogenesis in aged rats by measuring Nrf2,PGC-1α,and PGC-1α-downstream gene expression levels,oxidative balance status,mitochondrial function,and behavioral parameters.Next,we established D-gal-induced aging models respectively in wild-type and Nrf2-deficient mice,and a H₂O₂-induced SH-SY5Y cell model to investigate the potential molecular mechanisms in PTX-induced anti-aging effects.We found that PTX administration enhanced antioxidative capability and promoted mitochondrial biogenesis,which might be closely related to the up-regulation of Nrf2 and PGC-1αthrough cAMP-CREB pathway.Part one PTX reduces oxidative damage and mitochondrial dysfunction for improving age-related behavioral deficitsObjective:To explore the effect of PTX on age-related behavioral deficits by analyzing the oxidative balance and mitochondrial function parameters in the brain of aged rats.Methods:To study the effect of PTX on brain function of aged rats and determine the optimal dosage of PTX by analyzing the changes of ethology and oxidative balance parameters.And the effects of PTX treatment on mitochondrial function were studied by analyzing the changes of mitochondrial respiratory chain complex activity and ATP content in the brain of aged rats.Results:1.PTX treatment ameliorated motor behavior deficits in aged rats.Post hoc tests revealed that vertical activity,horizontal activity,and total path length were significantly decreased in the 24Mon group compared to the6Mon group（P<0.01）.All three behavioral parameters were significantly increased in the 24Mon-Ptx60（vertical activity and horizontal activity:P<0.05;total path length:P<0.01）and 24Mon-Ptx100 groups（horizontal activity:P<0.05;vertical activity and total path length:P<0.01）compared to the 24Mon group.2.PTX treatment improved cognitive function in aged rats.Post hoc analysis showed that the 24Mon group exhibited a longer escape latency to reach the platform（P<0.01）,fewer platform crossings（P<0.01）,and less time in the target quadrant（P<0.01）compared to the 6Mon group.Administration of 60 or 100 mg/kg of PTX shortened escape latency to reach the platform（P<0.01）and significantly increased the number of platform crossings（P<0.01）and time spent in the target quadrant（P<0.01）in aged rats.3.PTX treatment improved brain oxidative balance in aged rats.Post hoc tests revealed increased MDA levels and a decreased GSH/GSSG ratio in the SN and HIPP of the 24Mon group compared to the6Mon group（P<0.01）.In addition,MDA levels were reduced and the GSH/GSSG ratio was increased in the SN of the 24Mon-Ptx40,24Mon-Ptx60,and 24Mon-Ptx100 groups,as well as in the HIPP of the 24Mon-Ptx60 and24Mon-Ptx100 groups,compared to 24Mon rats（P<0.01）.4.PTX administration reduced mitochondrial dysfuction in the aged rat brain.4.1 PTX administration increased mitochondrial complex activity in the aged rat brain.Post hoc tests revealed a 32.23%reduction in complex I activity in the SN,as well as decreases in complex V activity of 22.4%in the SN and 31.0%in the HIPP,in the 24Mon group relative to the 6Mon group（P<0.01）.Additionally complex I activity in the SN increased by 21.1%（P<0.05）and complex V activity in the HIPP increased by 23.5%（P<0.05）in the24Mon-Ptx60 group compared to the 24Mon group.4.2 PTX administration increased mitochondrial ATP levels in the aged rat brain.Mitochondrial ATP levels were decreased by 31.5%in the SN and 28.6%in the HIPP of the 24Mon group relative to the 6Mon group（P<0.01）.Mitochondrial ATP levels were increased by 18.4%in the SN and 15.2%in the HIPP of the 24Mon-Ptx60 group compared to the 24Mon group（P<0.05）.Summary::1.PTX treatment ameliorated motor behavior and cognitive function deficits in aged rats.2.PTX treatment improved brain oxidative balance in aged rats.3.PTX administration reduced mitochondrial dysfuction in the aged rat brain.Part two PTX promotes Nrf2 activation and mitochondrial biogenesis in the brain of aged ratsObjective:To investigate the effects of PTX treatment on antioxidant capacity and mitochondrial biogenesis in the brain of aged rats.Methods:By analyzing the changes of Nrf2 activation,mitochondrial biogenesis related gene expression,mitochondrial content and cAMP content to study the mechanism of PTX treatment improving antioxidant capacity and mitochondrial dysfunction in the brain of aged rats.Results:1.PTX treatment increased Nrf2 levels in the SN and HIPP of aged rats.Nrf2 m RNA and intranuclear Nrf2 protein levels were lower in the SN and HIPP of 24Mon rats than in 6Mon rats（P<0.01）,while rats in the24Mon-Ptx60 group had higher Nrf2 m RNA and intranuclear Nrf2 protein levels in the SN and HIPP than rats in the 24Mon group（P<0.01）.2.PTX treatment upregulated PGC-1αand downstream gene expression in the aged rat brain.Post hoc analysis revealed that PGC-1α,NRF-1,and TFAM m RNA and protein expression was significantly downregulated in the 24Mon group compared to the 6Mon group（P<0.01）and upregulated in the 24Mon-Ptx60group compared to the 24Mon group（P<0.01）.3.PTX treatment increased mitochondrial content in the aged rat brain.CS activity and mt DNA copy number were reduced in the SN and HIPP of the 24Mon group compared to the 6Mon group（P<0.01）.In addition,CS activity and mt DNA copy number increased in the SN（P<0.01）and HIPP（CS,P<0.05;mt DNA copy number,P<0.01）of the 24Mon-Ptx60 group compared to the 24Mon group.4.PTX treatment increased cAMP content in the aged rat brain.cAMP content differed among the experimental groups in both the SN and HIPP（P<0.01）.cAMP content increased by 12%in the SN and 14.7%in the HIPP of the 24Mon-Ptx60 group compared to the 24Mon group（SN,P<0.01;HIPP,P<0.05）.Summary::1.PTX treatment increased Nrf2 levels in the SN and HIPP of aged rats.2.PTX treatment promoted mitochondrial biogenesis and increased mitochondrial content in the aged rat brain.3.PTX treatment increased cAMP content in the aged rat brain.Part three PTX enhances antioxidant capacity and mitochondrial biogenesis in D-galactose-induced aging mice by activating Nrf2/ARE pathwayObjective:To explore the molecular mechanism of PTX treatment on brain antioxidant capacity and mitochondrial biogenesis.Methods:We treated D-galactose（D-gal）-induced aging mice with PTX and measured changes in the degree of oxidative damage,mitochondrial content,nuclear factor erythroid 2-related factor 2（Nrf2）activation levels,and antioxidant and peroxisome proliferator activated receptor-gamma coactivator 1-alpha（PGC-1α）-dependent mitochondrial biogenesis gene expression.Results:1.Effects of PTX administration on oxidative balance status changes in D-gal-induced aging Nrf2-deficient mice brain.Post hoc test revealed that the percentage of karyopyknosis（except for CA3 region）,the AOD of 3-NT-IR cells,PCs content,and MDA levels were significantly elevatedin the KO-D-gal group compared to the KO-CON group（P<0.01）.Besides,the percentage of karyopyknosis in CA1 and DG regions,as well as the AOD of 3-NT-IR cells in CA1 region were reduced（P<0.01）in the KO-D-gal-PTX group compared to the KO-D-gal group,but not restored to the levels of the KO-CON group.PCs content and MDA levels showed a slight reduction in the KO-D-gal-PTX group without statistical significance.2.Effects of PTX administration on mitochondrial biogenesis in D-gal-induced aging Nrf2-deficient mice brain.The results of EM showed that the mitochondrial ultrastructure in WT-CON and KO-CON groups was normal.However,the mitochondria from the KO-D-gal group showed massive swelling with architectural disruption,which was consistent with the WT-D-gal group.The above abnormal ultrastructure alterations in mitochondria were ameliorated by PTX treatment to a certain content.Furthermore,mitochondrial number and size,as well as mt DNA copy number significantly differed among the experimental groups（P<0.01）.There were no significant differences between the WT-CON and KO-CON groups in the above indices.A reduction of mitochondrial number and mt DNA copy number,as well as an increase of mitochondrial size was detected in the KO-D-gal group over the KO-CON group（P<0.01）.After PTX administration,mitochondrial number and mt DNA copy number was enhanced but did not reach the normal level（P<0.01）,while no differences were observed in mitochondrial size.3.Effects of PTX administration on antioxidant related gene expression in D-gal-induced aging Nrf2-deficient mice brain.HO-1 and NQO1 protein levels were reduced in the KO-CON group following Nrf2 deficiency（P<0.01）.The results in the KO-D-gal group indicated that D-gal treatment up-regulated p16 and Ager m RNA levels（P<0.01）,down-regulated HO-1,NQO1,SOD2,CAT and GPx1 m RNA levels（P<0.01）,and reduced HO-1 and NQO1 protein levels（HO-1:P<0.05;NQO1:P<0.01）in the hippocampus compared to the KO-CON group.However,the m RNA expression of HO-1,NQO1,SOD2,CAT,and GPx1,as well as the protein expression of HO-1 and NQO1 were elevated to a small extent in the KO-D-gal-PTX group compared to the KO-D-gal group（CAT m RNA levels:P<0.05;others:no significant）.The p16 and Ager m RNA levels were reduced（P<0.05）in the KO-D-gal-PTX group compared to the KO-D-gal group.4.Effects of PTX administration on mitochondrial biogenesis related gene expression in D-gal-induced aging Nrf2-deficient mice brain.The results in the KO-D-gal group indicated that D-gal treatment reduced PGC-1α,NRF-1 and TFAM protein levels in the hippocampus compared to the KO-CON group（P<0.01）.The PGC-1α,NRF-1 and TFAM m RNA and protein levels were elevated in the KO-D-gal-PTX group compared to the KO-D-gal group（P<0.01）.Summary::1.PTX treatment improved the oxidative damage and the expression of antioxidant genes in brain of D-gal-induced aging Nrf2-deficient mice in some content.2.PTX treatment could mildly increase mitochondrial biogenesis in the brain of D-gal-induced aging Nrf2-deficient mice.3.PTX-induced,Nrf2-and PGC-1α-dependent increases in antioxidative capability and mitochondrial biogenesis might underlie the antiaging effects of PTX.Part four PTX increases Nrf2 and PGC-1αthrough cAMP-CREB pathwayObjective:To investigate the potential molecular mechanisms in PTX-induced anti-aging effects.Methods:We established the oxidative damage model of SH-SY5Y cells induced by hydrogen peroxide（H₂O₂）and knocked down CREB gene expression by si RNA interference to explore the molecular mechanisms of PTX-induced anti-aging effects.Results:1.Effects of PTX administration on H₂O₂-induced changes in SH-SY5Y cells.Cell viability was increased in the four PTX pretreatment groups compared to the H₂O₂ group（P<0.01）.Among the four PTX pretreatment groups,cell viability in the 1 m M PTX+H₂O₂ group was the highest（relative to 0.25 m M PTX:P<0.01,relative to 2 m M PTX:P<0.05）.In addition,we found that H₂O₂ treatment down-regulated p-CREB,p-Nrf2,and PGC-1αprotein levels compared to the control group（P<0.01）,whereas PTX elevated the expression of p-CREB,p-Nrf2,and HO-1 in the four PTX pretreatment groups compared to the H₂O₂ group（P<0.01）.Besides,except for the 0.25m M+H₂O₂ group,PGC-1αprotein levels were elevated in the other three PTX pretreatment groups relative to the H₂O₂ group（P<0.01）.Moreover,p-CREB and HO-1 protein levels were increased in the 1 m M PTX pretreatment group compared to the 0.5 m M or 2 m M PTX pretreatment groups（P<0.01）.The p-Nrf2 and PGC-1αprotein levels were increased in the0.5 m M and 1 m M PTX pretreatment groups compared to the 0.25 m M or 2m M PTX pretreatment groups（p-Nrf2:P<0.01,PGC-1α:P<0.05）.2.Effects of PTX administration on H₂O₂-induced changes in SH-SY5Y cells after si CREB.Compared with the NC group,CREB,p-Nrf2,and PGC-1αprotein levels in the NC-H₂O₂ group were reduced by 59.65%,64.28%,52.86%,respectively（P<0.01）,while the three protein levels were decreased by72.77%,64.95%,76.28%,respectively（P<0.01）,when the CREB gene was silenced.CREB,p-Nrf2,and PGC-1αprotein levels were elevated in the NC-H₂O₂-PTX group compared to the NC-H₂O₂ group（P<0.01）,which was consistent with the preceding findings.Except for a reduction of p-Nrf2expression in the si CREB-H₂O₂group relative to the si CREB group（P<0.05）,no significant differences were found among the si CREB,si CREB-H₂O₂,and si CREB-H₂O₂-PTX groups in the three protein expression.Summary:1.PTX pretreatment elevated cell viability and the expression of p-CREB,p-Nrf2,HO-1 and PGC-1αin H₂O₂-induced SH-SY5Y cells,and the protect effects of 1 m M PTX pretreatment was more effectively.2.CREB and Nrf2/PGC-1αact in a linear way,and the beneficial effects of PTX administration on H₂O₂-induced changes might depend on CREB pathway activation.Conclusion:1.PTX reduced oxidative damage and mitochondrial dysfunction for improving age-related behavioral deficits.2.PTX enhanced antioxidant capacity and promoted mitochondrial biogenesis by increasing Nrf2 and PGC-1α.3.PTX increased Nrf2 and PGC-1αthrough cAMP-CREB pathway.