Neuroplasticity-associated Protein Changes Related To Endoplasmic Reticulum Stress And Autophagy In The Prefrontal Cortex Of Obese Rats And The Regulatory Effects Of Aerobic Exercise

Objective:Animal and epidemiological studies have found that obesity not only harms the physiological functions of peripheral tissues and organs,but also affects the central nervous system?CNS?,which harms brain health and causes cognitive dysfunction.High-fat diet and obesity first lead to disorders of glucose and lipid metabolism in peripheral and central systems,affecting CNS signaling,causing neuronal apoptosis,impairing brain tissue structure and function,reducing cognitive function,and increasing the risk of neurodegeneration'diseases such as dementia.Therefore,obesity is an important risk factor for brain diseases.However,the causes of cognitive decline in obesity are still not fully understood,especially the prefrontal cortex,which is most closely related to cognitive function,still lacks relevant research.In this study,we observed changes in NMDAR1,N-AChR,SYN38,and BDNF in the prefrontal cortex of obese rats induced by high-fat diet,and whether endoplasmic reticulum stress,autophagy,and apoptosis occurred in the prefrontal cortex of obese rats,and whether the molecular signal changes.To analyze whether ERS,autophagy and apoptosis are the main reasons for the decreased expression of neuroplastic proteins.Furthermore,the role of endoplasmic reticulum stress and autophagy in affecting cognitive function in obese individuals was evaluated.The effect of aerobic exercise on the reversal of the above phenomenon in obese rats was observed.To investigate whether aerobic exercise can improve ERS,autophagy and apoptosis,and related signaling pathways in prefrontal lobes,and to explore the role of aerobic exercise interventions in improving ERS and promoting neuroplastic protein expression.Methods1^st part:150 male Sprague–Dawley?SD?rats were purchased from Shanghai Lab Animal Center?Certificate SCXK 2013–0016?at the age of 7 weeks?220g±10g?,and housed in the SPF animal research center of Shanghai University of Sport?SYXK2014-0002?.The rats had free access to water and diet.All studies were performed in accordance with the Science Research Ethics Committee of Shanghai University of Sport?No.2015013?.Experimental protocols were approved by the Animal Care and Use Committee at the Shanghai University of Sport.All efforts were made to minimize the number of animals involved and potential of sufferings.Animals were randomly divided into two groups with similar body weights?BW?,one was assigned to the high-fat diet?HFD,40%kcal from fat,n=110?,the other was assigned to normal chow diet?NCD,12.5%kcal from fat,n=40?.Animals were fed HFD for 8 weeks to promote excessive weight gain.BW was monitored once a week throughout the experiment.At the end of the 8th week,the BW value of NCD group was expressed as mean and standard deviation?SD?.HFD group animals that were greater than NCD group mean BW+1.4 SD were designated as diet-induced obesity?DIO?rats,according to the method that others have previously used.Then,NCD rats and DIO rats were divided into 4 groups by a randomized block design according to diet and exercise status:normal diet control sedentary group?CS,n=12?,normal diet with aerobic exercise group?CE,n=12?,obesity sedentary group?OS,n=12?,obesity with aerobic exercise group?OE,n=12?.DIO rats remained on HFD,and control rats on NCD until study completion.Rats in the exercise group underwent aerobic exercise for 8 weeks.At the end of the 17th week of the experiment,each group of rats was fasted for 12 hours overnight.After anesthesia was sacrificed,5 ml of blood was collected in the inferior vena cava of each rat,and the prefrontal lobe tissue was dissected.The protein was extracted with the protein extraction kit.Determination of triglyceride?TG?,total cholesterol?TC?,low-density lipoprotein cholesterol?LDL-C?and high-density lipoprotein cholesterol?HDL-C?in rat serum using a spectrophotometer,and the expression levels of related proteins in the prefrontal cortex of rats were detected by Western Blot methods.Including:FATP1?1:1000?,GRP78?1:1000?,PERK?1:1000?,phospho-PERK?1:1000?,IRE1??1:1000?,phosphor-IRE1??1:1000?,LC3?/??1:1000?,NMDAR1?1:2000?,AchR-7?1:2000?and synaptophysin?1:500?were used to detect membrane proteins.Bax?1:1000?,Bcl-2?1:1000?,caspase-12?1:2000?,eIF2??1:1000?,p-eIF2??1:1000?,Beclin-1?1:1000?,BDNF?1:1000?,PI3K?1:1000?,p-PI3K?1:1000?,Akt?1:1000?and p-Akt?1:1000?were also used to detect cytoplasmic proteins.PPAR??1:500?,CREB?1:1000?,phospho-CREB?Ser133??1:1000?,ATF4?1:1000?,p-JNK?1:1000?and CHOP?1:250?were used to detect nuclear proteins.Moreover,?-Actin?1:2000?,GAPDH?1:2000?,?-tubulin?1:2000?and Lamin B1?1:1000?was used as the loading control.2^nd part:SPF-class newborn SD rats?within 24 hours of birth?,male or female,were isolated from the prefrontal cells of newborn rats and cultured for 6 days.The high-fat medium was prepared with palmitic acid,and the ERS inhibitor?4-PBA?,autophagy inhibitor?3-MA?and PPAR?agonist?rosiglitazone?were added respectively.The prefrontal cells were further cultured for 24 hours.The growth state of the cells and the apoptosis rate were measured by flow cytometry to determine the optimal use concentration of the different media.The experiment was divided into five groups:?1?normal control group?control group?,use Neurobasal serum-free medium;?2?high-fat group?PA group?,Neurobasal medium supplemented with 0.5 mM palmitic acid;?3?ERS inhibitor group?4-PBA group?),5 mM 4-PBA was added to Neurobasal medium of 0.5 mM palmitic acid;?4?Autophagy inhibitor group?3-MA group?,2.5 mM 3-MA was added to Neurosal medium of 0.5 mM palmitic acid.?5?PPAR?agonist group?Rosi group?,40?m of rosiglitazone was added to Neurosal medium of 0.5 mM palmitic acid.The cells were cultured for 24 hours,the cells were collected,and the expression of related proteins in the prefrontal cells was examined by Western Blot.Results1^st part:1.After 8 weeks of high-fat diet,the weight of rats in high-fat diet group was significantly higher than that in normal diet group?p<0.01?,and 63 rats meeting obesity criteria.The success rate of obesity modeling was 57.27%?63/110?.Compared with the normal diet group,the concentrations of TG,TC and LDL-C in the serum of obese rats were significantly increased?p<0.01?,and the concentration of HDL-C was significantly decreased?p<0.01?.2.After 8 weeks of aerobic exercise intervention,the food intake and body weight of the OE group were significantly lower than those of the OS group?p<0.05,p<0.01?,and the concentrations of TG,TC and LDL-C were also significantly decreased?p<0.01?,HDL-C was significantly increased?p<0.01?in the serum.3.The protein expression of FATP1?F?1,20?=43.14,p<0.01?have significant interaction effects.Post-hoc results showed that the level of FATP1 in the OS group was significantly higher than that in the CS group?p<0.01?.After 8 weeks of aerobic exercise,the FATP1 content in the prefrontal lobe of the OE group was significantly lower than that of the OS group?p<0.01?.4.Compared with the CS group,the expression levels of GRP78?p<0.01?, p-PERK/PERK?p<0.01?,and p-eIF2?/eIF2??p<0.01?were significantly increased in the OS group;OE compared with OS,the expression of GRP78 ?p<0.01?,p-PERK/PERK?p<0.01?,p-eIF2?/eIF2??p<0.01?decreased significantly;OE compared with CS,the ratio of p-eIF2?/eIF2??p<0.05?was significant significantly increased.There was no significant difference in GRP78 and p-PERK/PERK indicators.Statistical results showed that p-IRE1?/IRE1??F ?1,20?=22.60,p<0.01?and ATF4?F?1,20?=4.582,p<0.05?had a significant dietary main effect,and as to ATF4?F?1,20?=69.47,p<0.01?,there is a significant main effect of motion,but there is no interaction.5.Beclin-1?F?1,20?=15.38,p<0.01?and LC3II/LC3I?F?1,20?=7.999,p<0.05?had significant main effects of diet,but no main effect of exercise,and there is also no interaction.6.Compared with the CS group,the levels of caspase12?p<0.01?,CHOP?p<0.01?,and Bax/Bcl-2?p<0.01?were significantly increased in the OS group;OE compared with the OS group,caspase12?p<0.01?.),CHOP?p<0.01?and Bax/Bcl-2?p<0.01?levels were significantly decreased;OE compared with CS,CHOP?p<0.01?and Bax/Bcl-2?p<0.05?levels were significant raise.7.Compared with CS group,the levels of BDNF?p<0.01?and SY38?p<0.01?in OS group were significantly lower.Compared with OS group,there was no significant difference in BDNF and SY38 expression in the OE group.However,AChR ?mainstream effect F?1,20?=21.99,p<0.01,main motor effect F?1,20?=13.69,p<0.01?had a major effect on diet and exercise,but no significant interaction.8.PPAR??dietary main effect F?1,20?=44.12,p<0.01,exercise main effect F?1,20?=11.99,p<0.01?,p-PI3K/PI3K?dietary main effect F?1,20?=15.23,p<0.01,exercise main effect F?1,20?=15.47,p<0.01?,caspase9?dietary maineffect F?1,20?=10.63,p<0.01,exercise main effect F?1,20?=6.208,p<0.05?and p-CREB/CREB?dietary main effect F?1,20?=154.8,p<0.01,exercise maineffect F?1,20?=233.9,p<0.05?had a major effect on diet and exercise,but nosignificant interaction.Compared with the CS group,the ratio of p-Akt/Akt ?p<0.01?was significantly decreased in the OS group.Compared with the OS group,the ratio of p-Akt/Akt?p<0.05?was significantly increased in the OE group.2^nd part:1.The addition of 0.5 mM PA to the cultured cells significantly induced apoptosis,and the apoptosis rate showed a dose-dependent effect curve.The cultured prefrontal cells were treated with different drugs.The apoptosis rate of PA group ?19.53±1.35?was significantly higher than that of C group?1.47±0.32,p<0.01?.Compared with PA group,5mM 4-PBA group?6.0±0.39?,20?M Rosi group ?11.8±0.97?and 40?M Rosi group?6.56±0.33?showed a significant decrease in apoptosis rate?p<0.01?,while 2.5mM 3-MA group?47.6±1.25?,5mM 3-MA group?60.5±4.25?and the 10 mM 4-PBA group?45.3±2.57?were significantly elevated?p<0.01?.2.Compared with the control group,the level of FATP1 was significantly increased in PA group?p<0.05?,and there was no difference in the level of FATP1 between 4-PBA,3-MA and Rosi compared with PA group.3.Compared with control group,the levels of GRP78?p<0.05?,p-IRE1?/IRE1?,p-PERK/PERK,p-eIF2?/eIF2?,ATF4,P-JNK,CHOP and caspase12 expression were significantly increased in PA group?p<0.01?.Compared with PA,the levels of GRP78?p<0.05?,p-IRE1?/IRE1??p<0.01?,p-PERK/PERK?p<0.05?,p-eIF2?/eIF2??p<0.01?,and ATF4?p?<0.01),p-JNK?p<0.01?,CHOP?p<0.01?,and caspase12?p<0.01?were significantly decreased in the 4-PBA group,the levels of GRP78?p<0.05?,p-PERK/PERK?p<0.05?and CHOP?p<0.01?in the 3-MA group were significantly increased,and the p-IRE1?/IRE1?ratio was significantly decreased?p<0.01?;the levels of GRP78?p<0.05?,p-IRE1?/IRE1? ?p<0.01?,ATF4?p<0.05?,p-JNK?p<0.05?,CHOP?p<0.05?,and caspase12 ?p<0.01?in the Rosi group were significantly reduced.4.Compared with the control group,the levels of Beclin-1 and LC3II/LC3I was significantly increased in the PA group?p<0.01?.Compared with the PA group,the levels of Beclin-1 and LC3II/LC3I in the 4-PBA group was significantly lower ?p<0.01?.The levels of Beclin-1 and LC3II/LC3I in the 3-MA group was significantly decreased?p<0.01?,while the autophagy index in the Rosi group was not significantly different.5.Compared with the control group,the ratio of p-PI3K/PI3K,p-Akt/Akt,and p-CREB/CREB was significantly lower in the PA group?p<0.01?,Bax/Bcl-2,Caspase9 was significantly elevated?p<0.01?.Compared with PA group,the ratio of p-CREB/CREB was significantly increased in the 4-PBA group?p<0.05?,the levels of PPAR??p<0.01?,p-PI3K/PI3K?p<0.05?and p-Akt/Akt ratio?p<0.01? was significantly decreased,and caspase9 was significantly increased in the 3-MA group?p<0.01?,the levels of PPAR?,p-PI3K/PI3K,p-Akt/Akt and p-CREB/CREB ratio were significantly elevated?p<0.01?,and the Bax/Bcl-2 ratio and caspase9 expression were significantly decreased in the Rosi group?p<0.01?.6.Compared with the control group,the levels of NMDAR1,SYN,BDNF and AchR in the PA group were significantly lower?p<0.01?.Compared with the PA group,the levels of SYN?p<0.05?and BDNF?p<0.01?were significantly more in the 4-PBA group,and the levels of SYN?p<0.05?,BDNF?p<0.01?and AChR?p<0.05? were significantly increased in the Rosi group,and there was no significant difference in the expression of each protein in the 3-MA group.Conclusions:1.In vivo studies have shown that high-fat diet can induce obesity in SD rats and lead to dyslipidemia,and promote expression of FATP1,and induce ERS and cause apoptosis in the prefrontal cortex of obese rats.in vivo and ex vivo Studies have shown that ERS inhibits the levels of PPAR?,p-PI3K,p-Akt and p-CREB,and these factors ultimately reduce the expression of neural plasticity-related proteins in the prefrontal cortex of obese rats.2.Aerobic exercise can improve dyslipidemia in obese rats,reduce the level of FATP1 in prefrontal cortex,alleviate ERS,decrease levels of apoptosis-related proteins,and promote levels of p-PI3K,p-Akt and p-CREB by activating PPAR? signaling pathway.Finally,the levels of neural plasticity-related proteins such as SYN,BDNF and AChR is increased.3.In vitro studies have shown that palmitic acid-induced prefrontal ERS is associated with increased levels of autophagy,and activation of autophagy inhibits ERS and its mediating apoptosis.Activation of PPAR?and its mediated signaling pathway by activators can attenuate ERS and its mediated apoptosis.PPAR? activation has protective effects on cells,and inhibition of ERS and activation of PPAR?signaling pathway can promote the levels of SYN,BDNF and AChR.