| Objective:Epidemiological and animal studies have demonstrated that high-fat diet induced obesity could decrease the expression of BDNF and synapses in hippocampus and impair hippocampal-dependent learning and memory ability, but their specific neurobiological mechanism has not been fully clarified. In recent years, many studies have found that high-fat diet or excess energy can induce peripheral tissues’ endoplasmic reticulum stress(ERS), and ERS will disrupt the functions of precursor protein processing and posttranslational modification to endoplasmic reticulum. Moreover, ERS also could affect other intracellular signaling pathways to regulate gene transcription in the cell nucleus. The biosynthesis of matured proteins will be influenced by the two aspects of ERS. But now it’s not clear that whether high-fat diet induced obesity in SD rats could cause hippocampal ERS, and whether hippocampal ERS could regulate neural plasticity protein expression. The purpose of this research was to study hippocampal ERS in vivo through establishing a high-fat diet induced obesity model of SD rats, and to explore the potential mechanism of ERS regulating the expression of hippocampal BDNF and SYN. Meanwhile, combined with the effect of aerobic exercise promoting neural plasticity protein expression, we wanted to discuss the regulating effect of aerobic exercise on hippocampal ERS and its possible mechanism. On this basis, high glucose and palmitic acid were added into basic culture medium to prepare intervention medium, which was used to culture hippocampal cells in vitro to imitate the symptoms of hyperglycemia and hyperlipidemia in obese rats. It’s aimed to study the induced effect of glucolipid metabolic abnormalities on hippocampal ERS, and to discuss the causal relationship between hippocampal ERS and biosynthesis of BDNF and SYN, as well as the specific regulatory mechanisms.Methods1st 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(SYXK 2014-0002). 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.The aerobic exercise training was performed as previously described with slight modifications. CE and OE animals were adapted to the treadmill for 5 days at 9th week, and the running speed was 11 m/min for 10 minutes at 0% grade on the first day and was gradually increased to 18 m/min for 40 minutes by the fifth day. The habituation week was followed by moderate intensity aerobic exercise training with the protocol of 18 m/min·d×40 min/d×5 days/week×8 weeks. All exercising rats completed the planned exercise training with no adverse events or major injuries.Whole body fat mass and fat percentage(fat mass/tissue mass) were assessed using dual energy X-ray absorptiometry and was performed after the last exercise training session(n=6/each group). Rats were anesthetized with an intraperitoneal injection of 10% chloral hydrate after 12-hour fast and then placed in prone position with extremities extended to enable full scanning of the radius and ulna. The first blood samples(n=12/each group) were collected from eye angular vein after 12 hours of fasting when the rats were divided into 4 groups at the 9th week. The second blood samples(n=12/each group) were collected from the abdominal aorta by laparotomy under the intraperitoneal injection anesthesia of 10% chloral hydrate after 8 weeks of exercise training. The samples were centrifuged(3000 rpm for 10 min, 4 °C) to collect serum, and stored at-80 °C until analysis. The serum was assayed for blood glucose(BG), triglyceride(TG), total cholesterol(TC), low-density lipoprotein cholesterol(LDL-C) and high-density lipoprotein cholesterol(HDL-C) with commercially available test kits.After 8 weeks of exercise training, samples of 6 rats per group were decapitated under the injection anesthesia of 10% chloral hydrate, and the bilateral hippocampi were quickly separated from the brain and frozen in liquid nitrogen. Membrane Nuclear and Cytoplasmic Protein Extraction kit was used to extract hippocampal proteins. Protein concentration was determined by the BCA method. For the protein assay, protein samples containing an equal amount of protein(30 mg) were electrophoresed on 8% or 10% SDS-PAGE gels and transferred to PVDF membranes. The membranes were blocked with 5% non-fat milk powder in TBST buffer and incubated overnight at 4°C with different primary antibodies. GLUT3(1:2000), FATP1(1:1000), PERK(1:1000), phospho-PERK(1:1000), IRE1α(1:1000), phosphor-IRE1α(1:1000) were used to detect membrane proteins, and β-Actin(1:1000) was used as the loading control. Bax(1:1000), Bcl-2(1:1000), and caspase-12(1:2000) were also used to detect membrane proteins, and GAPDH(1:2000) was used as the loading control. GRP78, e IF2α and phospho-e IF2α(Ser51), with dilution of 1:1 000, and Heme Oxygenase 1(HO-1) with dilution of 1:1000, were used to detect cytoplasmic proteins. And β-Actin(1:1000) was used as the loading control. The following primary antibodies were also used to detect cytoplasmic proteins, pro BDNF(1:100), BDNF(1:1000), synaptophysin(1:5000), IL-1 beta(1:5000); p38 MAPK, phospho-p38MAPK(Thr180/Tyr182), Erk1/2, phospho-Erk1/2(Thr202/Tyr204) with dilution of 1:1000 and NLRP3/NALP3(1:250). And GAPDH(1:2000) was used as the loading control. Moreover, CREB(1:1000), phospho-CREB(Ser133)(1:1000), Nrf2(1:1000), CHOP(1:250) were used to detect nuclear proteins, and Lamin B1(1:1000) was used as the loading control. After rinsing with TBST, the membranes were incubated with peroxidase-conjugated affinipure goat anti-rabbit Ig G(H+L)(1:5000) for 1h at RT. To visualize the immunoreactive proteins bands, an ECL kit was used according to the manufacturer’s instructions. The density of each band was quantified using the Gel Doc XR system. The data were analyzed and presented as the relative density of each protein relative to loading controls, and phospho-protein relative to total protein.2nd part:Newborn SD rats were decapitated to isolate the hippocampi, and culture hippocampal cells 5 days with Neurobasal culture medium containing 2% B27 and 1% penicillin-streptomycin(basic culture medium). D-glucose and palmitic acid(PA) were added into basic culture medium to prepare “high glucose and PA culture medium”, and a certain concentration of ERS inhibitor of 4-PBA or Nrf2 activator of TBHQ was added into high glucose and PA culture medium. Different types of culture medium were separately used to treat matured hippocampal cells. Flow Cytometer was used to detect cell viability to choose the best culture medium concentration and treated time. After that, the experiment was divided into four groups:(1) basic culture medium group(control group),(2) high glucose and PA culture medium group(the final concentration of 50 m M D-glucose and 500 μM PA were added into basic culture medium),(3) ERS inhibitor group(the final concentration of 20 m M inhibitor 4-PBA was added into basic culture medium),(4) Nrf2 activator group(the final concentration of 100 μM activator TBHQ was added into basic culture medium). Different types of culture medium were separately used to treat matured hippocampal cells for 6 h. RT-PCR was used to detect BDNF m RNA, SYN m RNA, NLRP3 m RNA and HO-1 m RNA. Western Blot was used to detect the related proteins in 1st part.Results1st part:1. The HFD group rats gained more body weight than NCD group after 8 weeks of high fat diet(p<0.01), and there were 63 rats meeting the obese criterion. There were higher levels of BG, TG, LDL-C(p<0.01), and lower levels of HDL-C(p<0.01) in obesity rats than those in NCD rats. 2. After 8 weeks of aerobic exercise intervention, there were less food intake, energy intake, body weight, whole fat mass, and fat mass/weight in OE group than those in OS group(p<0.05,p<0.01). Meanwhile, OE group gained less BG, TG, TC, LDL-C, and more HDL-C than OS group(p<0.01). 3. There were higher levels of hippocampal GLUT3 and FATP1 in OS group than those in CS group(p<0.01). However, the levels of hippocampal GLUT3 and FATP1 were significantly decreased in OE group than those in OS group(p<0.01), after aerobic exercise intervention. Furthermore, compared with CS group, the levels of GLUT3 were significantly increased in OE group(p<0.01), but there were no significant differences about FATP1 levels. 4. OS group not only had more GRP78, p-PERK/PERK, p-IRE1α/IRE1α and p-e IF2α/e IF2α than CS group(p<0.01), but also had more caspase12, CHOP, Bax(p<0.01), and less Bcl2(p<0.01), which suggesting hippocampal ERS was obviously induced in OS group. Compared with OS group, OE group had less ERS marker proteins and ERS-induced apoptotic proteins(p<0.01). 5. There were lower levels of hippocampal pro BDNF, BDNF and SYN in OS group than those in CS group(p<0.01). However, these neuronal plasticity proteins were significantly increased in OE group than those in OS group(p<0.01), and compared with CS group, CE group had the similar changes(p<0.05). 6. Compared with CS group, there were lower levels of hippocampal p-p38/p38, p-ERK/ERK, p-CREB/CREB(p<0.01), and higher levels of hippocampal NLRP3 and IL-1β(p<0.01) in OS group. But the levels of p-p38/p38, p-ERK/ERK,p-CREB/CREB were significantly elevated(p<0.01), and the levels of NLRP3 and IL-1β were obviously decreased(p<0.01) in OE group than those in OS group. Moreover, CE group had more p-p38/p38, p-ERK/ERK, p-CREB/CREB(p<0.05), and less NLRP3 and IL-1β(p<0.05) than CS group. 7. OS group had less levels of hippocampal Nrf2 and HO-1 than CS group(p<0.01), but after aerobic exercise intervention, the two proteins levels were markedly enhanced in OE group than those in OS group(p<0.01). Compared with CS group, CE group had more levels of Nrf2 and HO-1(p<0.01).2nd part:1. The levels of hippocampal GLUT3 and FATP1 were significantly increased in high glucose and PA culture medium group than those in basic culture group(p<0.01). When used ERS inhibitor or Nrf2 activator in high glucose and PA culture medium, there were no significant changes in the two proteins compared to high glucose and PA culture medium. 2. Compared with basic culture medium group, there were more GRP78, p-PERK, p-IRE1α and p-e IF2α(p<0.01), more caspase-12, CHOP and Bax(p<0.01), and less Bcl-2(p<0.01) in high glucose and PA culture medium group. ERS inhibitor group and Nrf2 activator group had less ERS marker proteins(p<0.01) and ERS-induced apoptotic proteins(p<0.01) than basic culture medium, except the Bax had no significant change in Nrf2 activator group than that in basic culture medium. 3. Compared with basic culture medium group, high glucose and PA culture medium group had less levels of pro BDNF, BDNF and SYN(p<0.01). But the three proteins levels were markedly increased both in ERS inhibitor group and Nrf2 activator group(p<0.01). 4. High glucose and PA culture medium group had less levels of p-p38, p-ERK, p-CREB, BDNF m RNA and SYN m RNA than basic culture medium(p<0.01). However, the phosphorylated levels of the three proteins and the expression of BDNF m RNA and SYN m RNA were obviously increased both in ERS inhibitorgroup(p<0.01) and in Nrf2 activator group(p<0.01). 5. Compared with basic culture medium group, high glucose and PA culture medium group had more NLRP3 and IL-1β(p<0.05,p<0.01), and the expression of NLRP3 m RNA was significantly enhanced(p<0.01). But the two inflammatory proteins and NLRP3 m RNA expression were markedly decreased both in ERS inhibitor group(p<0.01) and in Nrf2 activator group(p<0.01). 6. The proteins levels of Nrf2 and HO-1, and the expression of HO-1 m RNA were significantly decreased in high glucose and PA culture medium group than those in basic culture medium(p<0.01). However, the two proteins and HO-1 m RNA expression had no significant changes in ERS inhibitor group, but there were markedly enhanced in Nrf2 activator group(p<0.01), both compared with basic culture medium.Conclusions:1. 8 weeks of high fat diet could make SD rats become obesity, which appeared the symptom of glucose and lipid metabolism disorders. Hyperglycemia and hyperlipidemia could promote the expression of hippocampal GLUT3 and FATP1. 2. High fat-diet induced obesity could cause hippocampal ERS, which could not only disturb endoplasmic reticulum functions, but also inhibit hippocampal p38/ERK-CREB signaling pathways, activate NLRP3-IL-1β signaling pathways and cause apoptosis, leading to decreased BDNF and SYN levels. 3. 8 weeks of aerobic exercise treadmill intervention could significantly alleviate glucose and lipid metabolism disorders, decrease GLUT3 and FATP1 protein expression, activate hippocampal Nrf2-HO-1 signaling pathways leading to decreased hippocampal ERS and apoptosis and increased BDNF and SYN levels. |
PDF Full Text Request