Ming-Zhe Qin, Xiang Zhou, Meng-Da Xu, Hao Wu, Bi-Xi Li, Guo-Sheng Gan, Ting Yu, Xiao-Yang Song

Department of Anesthesiology, Central Theater General Hospital of Chinese People's Liberation Army, Wuhan 430074, Hubei, China

Keywords:

ABSTRACT

1. Introduction

Postoperative cognitive dysfunction(POCD) refers to patients without mental disorders before surgery, due to a variety of factors leading to brain dysfunction, resulting in reversible acute mental disorder syndrome after surgery [1].The use of general anesthesia may be an important factor leading to POCD [2]. Studies have reported that the expression of β-amyloid protein in peripheral blood of elderly patients after general anesthesia is significantly higher than that of young and middle-aged patients. In addition, the expression of Aβ in peripheral blood of elderly patients is significantly up-regulated after surgery [3], while in Alzheimer's A large amount of Aβ accumulation can also be seen in the patient's brain [4], so POCD caused by anesthesia may be similar to Alzheimer's. POCD is similar to AD in pathogenesis, clinical manifestations, and morphological and pathological changes in the central nervous system, so AD treatment drugs may be potential POCD treatment drugs. Rapamycin is a macrolide compound with anti-tumor [5], anti-aging [6] activities. The latest research shows that rapamycin can improve the cognitive function of AD mice [7], delay the aging process, anti-oxidation, anti-inflammation and inhibition of tau protein hyperphosphorylation and Aβ production and aggregation. Rapamycin is a potential AD treatment drugs [8]. Does rapamycin also improve the cognition of patients with POCD Features? Therefore, in this study, a post-anesthesia POCD model was established to investigate the effect of rapamycin on the cognitive function of POCD rats and explore its related mechanisms.

2.Materials and methods

2.1 Animals

100 Wistar rats, half male and half female, 20 weeks old, were purchased from Hubei experimental animal research center, Certificate No.:SCXK(E)2003-0005. The rats were kept adaptively for one week in an animal room with a room temperature of 25℃ and a humidity of 45%, , drinking water freely and eating standard food.

2.2 Main reagents and instruments

Ketamine, rapamycin, 3-methyladenine and everolimus were all purchased from sigma Aldrich reagent company. β- actin, P13K, p-Akt, p-mTOR, 4ebp1 and β - actin antibodies were all purchased from cell signaling technology reagent company ; Goat anti mouse second antibody (sigma Aldrich reagent company); Ferritin H, ferritin L, S100 β, Beclin1 and ATG5 ELISA kits were purchased from Shanghai Yansheng Industry Co., Ltd; Morriss water maze (Shanghai Xinruan Information Technology Co., Ltd.); SDS-PAGE electrophoresis tank (Bio-Rad Life Medical Products Co., Ltd.); Microplate reader (Thermo scientific company); TANON 5500 automatic chemiluminescence imaging analysis system (Shanghai Tanan Technology Co., Ltd.).

2.3 Methods

All rats were adapted to water maze with water temperature of 24℃ for 2 minutes and trained for 5 days. 80 rats were intraperitoneally injected with 70 mg / kg ketamine once a day for 7 days to establish a post-anesthesia POCD model. 20 blank control group were injected with equal volume of normal saline. 80 model rats were randomly divided into model group, rapamycin group, everolimus positive group, 3-methyladenine group, 20 rats in each group. The model group was intraperitoneally injected with the same amount of normal saline, the rapamycin group was intraperitoneally injected with 3.5 mg / kg rapamycin, the everolimus positive group was intraperitoneally injected with 5.0 mg / k everolimus, and the 3-methyladenine group was intraperitoneally injected with 3.5mg / kg rapamycin + 2.0mg / kg 3-methyladenine. Continuous 7d.

Morris water maze was used to measure the retention time, the number of crossing platform and the latency time of working memory in each group [9]. All rats performed space-acquisition experiments first. The rats were placed in the second quadrant, third quadrant, and fourth quadrant. The time when the rats entered the hidden escape platform in the first quadrant was observed. They were trained 3 times a day for 5 consecutive days. Withdraw the hidden escape platform in the first quadrant, and record the retention time of the rat in the first quadrant and the number of crosses the platform within 60 seconds. The retention time and the number of crosses the platform were used to evaluate the spatial memory of the rat.Placed the hidden escape platform in any quadrant, and put the rats into the water maze from other quadrants. After the rats successfully boarded the platform, put the rats back in the same place, and recorded the time when the rats re-boarded the platform as the latency time of working memory. The the latency time of working memory is used to evaluate the short-term working memory of rats.

After the last administration, 10 rats were drawn from each group, anesthetized with pentobarbital, decapitated, the brain was dissected, and various brain tissues were separated. After homogenizing the hippocampal tissue, centrifuge at 14000 rpm for 10 min, separate the supernatant, and store at -80 ℃ . The concentration of ferritin H (Ferritin H), ferritin L (Ferritin L), S100β, and Beclin1 was measured using the Elisa assay kit. Western blot method was used to detect P13K, p-Akt, p-mTOR, 4EBP1 protein, SDS-PAGE, membrane transfer, blocking, incubation of first antibody overnight, second antibody incubation for 1h, chemiluminescence, photograph and analysis.

2.4 Statistics and analysis

SPSS13.0 statistical software was used. The analysis of variance was used in the comparison among groups, if there were statistical differences, the t-test was used to compare each other. P < 0.01 has significant difference, P < 0.05 has statistical significance.

3.Resluts

3.1 Effect of rapamycin on the cognitive function of POCD rats

The retention time and the number of crossing platform in the Rapamycin group and positive group were significantly higher than in the model group(t=6.645，P＜0.01；t=9.60，P＜0.01；t=5.961，P＜0.01；t=4.553，P＜0.01), the latency time of working memory was significantly lower than in the model group(t=3.799，P＜0.01；t=7.303，P＜0.01). The retention time and the number of times of crossing platform in the 3-methyladenine group were significantly lower than in the Rapamycin group(t=5.138，P＜0.01；t=4.437，P＜0.01), the latency time of working memory was significantly higher than in the Rapamycin group(t=2.808，P＜0.05). Table 1.

3.2 Effects of rapamycin on cognitive function related proteins in POCD rats

Ferritin H, ferritin L and S100 β in rapamycin group and positive group were significantly lower than those in model group(t=3.248，P＜0.01；t=3.021，P＜0.01；t=4.137，P＜0.01；t=4.906，P＜0.01；t=2.915，P＜0.01；t=2.900，P＜0.01). Fserritin H, ferritin L and S100 β in 3-methyladenine group were significantly higher than those in pamamycin group(t=2.247，P＜0.05；t=3.330，P＜0.01；t=2.119，P＜0.05).Table 2.

Table 2 Effects of rapamycin on cognitive function related proteins in POCD rats(x±s， n=10)

3.3 Effects of rapamycin on autophagy related proteins in POCD rats

Beclin1 and ATG5 in rapamycin group and positive group were significantly higher than those in model group(t=7.293，P＜0.01；t=10.903，P＜0.01；t=10.118，P＜0.01；t=8.062，P＜0.01). Beclin1 and ATG5 in 3-methyladenine group were significantly lower than those in pamamycin group(t=5.071，P＜0.05；t=8.663，P＜0.05). Table 3.

Table 3 Effects of rapamycin on autophagy related proteins in POCD rats（x±s， n=10）

3.4 Effect of rapamycin on P13K / mTOR signaling pathway

After POCD model was established, P13K, p-Akt, p-mTOR and p-4ebp1 protein were up-regulated. After administration of rapamycin and positive drug, the protein levels of P13K and p-Aktdid not change, the protein levels of p-mTOR and P-4EBP1 were down regulated, and the protein levels of p-mTOR and P-4EBP1 were up regulated after using 3-methyladenine.

Table 1 Effect of rapamycin on the cognitive function of POCD rats（x±s， n=10）

Figure 1 Effect of rapamycin on P13K / mTOR signaling pathway

4.Discussion

POCD is a common central nervous system complication after anesthesia, especially in the elderly. POCD patients' memory, attention, orientation, self-control decreased, extended recovery time. In severe cases, patients may have permanent cognitive dysfunction, or even death [10]. At present, the research on POCD is not perfect. The pathogenesis of POCD may be related to age, type of operation, using general anesthesia drugs, stress, activation of inflammatory response and other factors [11]. The current POCD model wass mainly based on sham operation [12], but the model ignored the role of general anesthesia drugs in the pathogenesis of POCD. In the previous study, we established a POCD model caused by repeated anesthesia. The model rats' staying time, the latency time of working memory increased, and the number of crossing the table decreased significantly, which indicated that repeated anesthesia could successfully establish a POCD model.

Rapamycin is a macrolide extracted from Streptomyces hygroscopicus. Rapamycin has a wide range of activities, such as antifungal [13], immunosuppressive, antitumor, anti-aging and so on. Caccamo et al. Showed that rapamycin, as a macrolide drug, could significantly improved the cognitive dysfunction of AD model mice, and its mechanism may be related to the inhibition of Aβ accumulation and tau protein hyperphosphorylation [14], and Aβ accumulation could also be seen in POCD rats, so rapamycin had the potential to treat POCD. Our study show that rapamycin could significantly improve the cognitive function of POCD rats. The retention time and the latency time of working memory of the model rats were reduced, and the number of crossing platform was significantly increased. Surgical anesthesia could lead to abnormal iron metabolism in the body. Hydroxyl free radicals catalyzed by free iron could react with lipid membranes and DNA to cause cell damage and lead to premature cell death. Abnormal brain iron metabolism is one of the initial causes of neuronal death in certain neurodegenerative diseases[15]. S100 β protein is involved in learning and memory function, which is highly correlated with POCD [16]. We found that rapamycin could reduced the contents of ferritin H, ferritin L and S100 β in the hippocampus of POCD rats. Through the whole animal experiment and molecular index, we confirmed our hypothesis: rapamycin could significantly improved the cognitive function of POCD rats, but how does rapamycin played this function?

Autophagy is a kind of internal phenomenon in cells, which is mainly used to regulate "long life" proteins and some organelle functions. A large number of evidences show that the function of autophagy lysosomal system gradually weakens with age, which may be the main mechanism of abnormal protein deposition in the process of individual degeneration [17-18]. Cai et al believe that the upregulation of autophagy activity plays an important role in improving neurodegenerative lesions[19]. Rapamycin is an important inhibitor of autophagy. Rapamycin may improve POCD by inhibiting autophagy activity. Therefore, we investigated the expression of Beclin1 and ATG5 protein in hippocampus of rats with POCD. The results showed that Beclin1 and ATG5 protein were significantly increased, rapamycin could improve the autophagy ability of rats with POCD and improve their cognitive function. The upstream pathway of rapamycin is the PI3K / Akt pathway [20], but the study results show that rapamycin had no effect on both. In the downstream, rapamycin could significantly reduced P-mTOR and P-4EBP1 proteins, and this process could be reversed by the autophagy inhibitor 3-methyladenine. The above results show that rapamycin could promoted autophagy in hippocampal tissue by affecting mTOR signaling pathway.

In conclusion, rapamycin can improve the cognitive function of POCD rats induced by anesthesia, and its mechanism may be related to the activation of mTOR signaling pathway by rapamycin to promote autophagy. This study further expands the clinical application of rapamycin, and provides a potential therapeutic drug for clinical POCD treatment.