2019 Volume 42 Issue 11 Pages 1867-1876
Anandamide (AEA) played potent neuroprotective activities via cannabinoid type 1 (CB1) and 2 (CB2) receptor. N-Linoleyltyrosine (NITyr), as an AEA analogue, was synthesized in our laboratory and evaluated the neuroprotective effects and mechanisms for the first time. NITyr was synthesized via substitution reaction. The neuroprotective effects of NITyr were evaluated in a gerbil model of transient cerebral ischemia. Each gerbil was subjected to open field test (OFT), Rotard rod test (RRT), Morris water maze (MWM) successively and executed after animal behaviors. Part of the brain was stained with hematoxylin and eosin (HE) and Nissl staining, and the rest for biochemical analysis. NITyr could not increase spontaneous locomotor activity and ameliorate the anxiety behavior in the OFT but could improve the motor coordination in the RRT and the spatial memory impairment in the MWM. Immunohistochemically, NITyr could attenuate the ischemia-induced neural loss in the hippocampus. The Enzyme-linked immunosorbent assay (ELISA) suggested that NITyr ameliorated the inflammation and oxidative stress. Consistently, NITyr could up-regulate the expressions of p-phosphadylinositol 3-kinase (PI3K) and p-Akt but not PI3K and Akt in the hippocampus. In addition to oxidative stress, CB2 receptor antagonist AM630 but not CB1 receptor antagonist AM251 could reverse the above phenomena. However, CB1 receptor antagonist AM251 could reverse oxidative stress. Accordingly, NITyr could up-regulate the expressions of CB2 but not CB1. NITyr could improve the motor coordination, learning and memory impairments, neural loss in the hippocampus and the inflammation of the mice via CB2 receptor involvement of PI3K/Akt signaling pathway.
Cerebral ischemia, an acute nerve injury caused by interruption of blood supply of the brain has become a primary threat to human health.1,2) Experimental evidence indicates that insufficient blood supply trigger multiple pathological processes such as oxidative stress, inflammation etc., which eventually lead to neuronal loss and progressive cognitive impairment, anxiety and behavioral deterioration. Although many neuroprotective agents protected against neuronal damage in cerebrovascular diseases, few neuroprotective agents are effective in vivo due to neglecting the correlation of multiple biological injuries of ischemia.3–5) Therefore, it became a hot topic about novel neuroprotective agents in ischemia via intervening in multiple pathways.
Recent evidences suggest that anandamide (AEA) plays crucial roles in neurodegenerative disease via cannabinoid type 1 (CB1) and 2 (CB2) receptors, which are correlation with oxidative and inflammatory response.6) However, the metabolism of AEA was rapid in vivo, only in a few minutes.7) Accordingly, AEA could not be used as a neuroprotective agent directly. Thus, the design, synthesis and initial biological evaluation of AEA analogues are ongoing research in our lab. In the previous studies, N-stearoyltyrosine developed in our laboratory was closely similar with AEA not only in structure but also in neuroprotective activities.8,9) However, the concentration of N-stearoyltyrosine must be higher several orders of magnitude than AEA to show good activity.10) Meanwhile, the activities of long-chain fatty acids with unsaturated bonds could be better than that of long-chain fatty acids with saturated bonds.11) Therefore, it is of great significance to synthesize unsaturated fatty acyl amino acids.
It has been reported that linoleic acid is good stability and has a wide range of pharmacological activities.11) Thus, linoleic acid was chose as the mother chain of unsaturated fatty acyl amino acids. Considering that N-stearoyltyrosine showed the best activities among a series of N-stearoyl amino acids.10) Tyrosine was chose as the binding chain of unsaturated fatty acyl amino acids. Based on the above researches, we synthesized AEA analogues-N-linoleyltyrosine (NITyr).
In the current study, the therapeutic activities and mechanisms of NITyr in the cerebral ischemia in gerbils were first investigated. Firstly, the behavioral, pathological characteristics and processes of mice were explored to assess the activities of NITyr. Next, the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway was studied to preliminarily confirm the possible mechanism of NITyr, in which CB2 receptor might be involved. It is hoped to find a novel neuroprotective agent.
Chemicals were purchased from China National Pharmaceutical Group Corporation (SINOPHARM, Beijing, China). The processed of reactions were monitored by silica gel (200–300 mesh ASTM). 1H- and 13C-NMR spectra were recorded at 400 and 100 MHz on a Bruker AM-400 spectrometer in chloroform-d solutions, respectively. Chemical shifts were reported in ppm (δ) and coupling constants (J) are in hertz (Hz). Mass spectra were recorded on an Agilent 1100 series LC/MSD chromatographic system (U.S.A.). The antibodies of PI3K, P-Akt, Akt, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin were purchased from Cell Signaling Technology (Shanghai, China). The antibodies of CB1 and CB2 were purchased from Abcam (Shanghai, China).
The Preparation of Chemical CompoundNITyr was synthesized using the following steps.12) Synthetic routes of NITyr (Chart 1): To a solution of linoleic acid in 10 mL ethyl acetate (EtOAc), N-hydroxysuccinimide and dicyclohexyl carbodiimide were added in 5 mL EtOAc in turn and the reaction was stirred for 48 h. TLC was used to confirm whether the reaction was completed. After the reaction was finished, the mixture was filtered, and the filtrate was collected and extracted with EtOAc (2 × 30 mL). The organic phase solution was condensed under decompression. The residue obtained was used for the next reaction as well as no further purification. Next the obtained residue (7.14 mol), tyrosine in 40 mL of 65% aq ethanol and triethylamine was mixed and the reaction was stirred for 72 h at room temperature. Then the reaction mixture was condensed under decompression, taken up in 25 mL EtOAc and sequentially washed with ice cold 1 (N) HCl (30 mL × 2) and saturated brine solution (25 mL × 2). The desired products were obtained by collecting the above organic phase which was collected, dried and evaporated under reduced pressure. The residue was purified by silica gel chromatography with chloroform–methanol (10 : 1) as the eluent. The product was obtained as a light-yellow syrup.
Male 12-week-old Mongolian gerbils (Experimental Animal Center, China) weighing 50–70 g were fed in a controlled environment at 24 ± 1°C for 5 d with a 12 : 12 h light–dark cycle. They had free access to get food and water. The experimental procedures were conducted in accordance with institutional guidelines for Animal Experimentation of Chengdu Medical College.
Drug TreatmentsThere are ten experimental groups (n = 10 each group): the sham group, the ischemic group, three ischemia groups treated with 1, 5 and 10 mg/kg NITyr, respectively, four ischemia groups treated with CB1 receptor antagonist AM251 (1 mg/kg), CB2 receptor antagonist AM630 (1 mg/kg), NITyr combined with AM251 and NITyr combined with AM630, respectively, one ischemia group treated with Nimodipine (10 mg/kg) as a positive control group. The drugs were dissolved in 0.1% dimethyl sulfoxide (DMSO) diluted with saline, and then the sham and ischemia group received an equal volume of 0.1% DMSO in saline. The experimental design was as follows (Chart 2): Each gerbil was given intraperitoneally in a volume of 0.1 mL per 10 g weight at 12, 24, 48, 72, 96 and 120 h after ischemia reperfusion. Gerbils were subjected to open field test (OFT) on the sixth day, the Rotarod test (RRT) on the seventh day and Morris water maze (MWM) on the twelfth day and executed on the seventeenth day after ischemia. After the animal experiment, the brain was removed from the skull. Some of the brains were used for histological examination and the rest of the brain for biochemical detection.
The model of ischemia–reperfusion caused by occlusion of the common carotid arteries was carried out according to the following procedures. All gerbils were anesthetized with pentobarbital, placed in the dorsal position, and then a middle vertical incision was made in the neck. Meanwhile, the bilateral carotid arteries were exposure, separation from the sympathetic nerves and adjacent veins, and then occlusion for 5 min with a nontraumatic micro aneurysm clip. The blood reperfusion was confirmed after the clip was removed. Blood flow was reconstructed by direct observation and then the wound was sutured by 3–4 loose silk stitches. The same operated animals without the carotid arteries occlusion were served as the sham group. Post-ischemic temperature was carefully monitored. The rectal temperature of the animals was maintained at 37.0 ± 0.5°C throughout the whole process of experiment and an incandescent lamp was used until the animal recovered consciousness.
Preparation of Brain Tissue SamplesFor histological examination, the brain tissue of gerbils was irrigated with saline and 4% paraformaldehyde in 0.1 M phosphate buffer solution (PBS) respectively and sequentially. Then, the brain was removed from the skull, post-fixed for at least 24 h in 4% paraformaldehyde, after that orderly dehydrated in PBS containing 10, 20 and 30% sucrose. The obtained brain was embedded in paraffin, cut into 5-µm-thick slices on the coronal plane serially and mounted on coated slides. For biochemical analysis, the brain was removed from the skull to 0.9% saline carefully and quickly, and the hippocampus of the brain was immediately separated, weighed and homogenized with a cold RIPA buffer solution (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) with protease inhibitors and phosphatase inhibitors (Apexbio, Beijing, China). The homogenates were centrifuged at 15000 × g for 10 min at 4°C, and then the supernatants were collected and stored at −70°C until used.
OFTThe locomotor activity of the gerbil was studied in the OFT as described in the previous study.13)
RRTThe rotarod performance of the mice was studied in the RRT. Before each experiment, the gerbil was placed on the stationary rotarod for 30 s. Then the animals were given 3 trials per day for 5 consecutive days on the rod (30 rpm over 2 min). The latency to fall was recorded for each trial.
MWMThe MWM test was begun on the seventeenth day after surgery to evaluate the learning and memory ability of gerbil. The apparatus was made up of a circular pool which is filled with water and surrounded by curtains to avoid environmental interference. Meanwhile, the escape platform, about 1.5 cm below the water surface, was placed in the middle of the target quadrant. During five consecutive days of training, each gerbil was practiced four times per day. During every training, the gerbil that faced the wall was placed in the pool randomly and allowed 60 s to find the escape platform. If the gerbil did not find the platform, it was guided to the platform by the experimenter and rested on the platform for 30 s to familiarize with the environment. On the sixth day of the training, the platform in the target quadrant was removed and the probe trials were conducted. Meanwhile, the animals were allowed to explore in the target quadrant for 60 s in which the platform was located previously. The time spent in the target quadrant and the escape latency of the gerbil were recorded through a video tracking system.
Histological ExaminationThe brain tissues were embedded, cut into 1–2 µm thick sections and stained by hematoxylin and eosin (HE) staining and Nissl staining via a routing staining protocol. The number of cells in the CA1 region was observed by microscope in a high-power optical microscope (400 × magnification). The cells with clear nucleus and nucleolus were optimally observed. The images were taken by optical microscope (Olympus, Tokyo, Japan).
Western Blot AnalysisThe tissue supernatants were collected, and the concentrations of total protein were subsequently evaluated by Bradford protein assay kit (Thermo Scientific, Shanghai, China). The protein samples were denatured at 99°C for 8 min, separated on 10% sodium dodecyl sulfate-polyacrylamide gel and successively electro-transferred onto a nitrocellulose membrane (Millipore, Shanghai, China). The nitrocellulose membranes were blocked in 5% non-fat milk powder in Tris-buffered saline containing 0.05% Tween-20 (TBST) at room temperature and then incubated overnight at 4°C with the following primary antibodies: rabbit anti-CB1 (1 : 2000) and anti-CB2 (1 : 1000); mouse anti-PI3K (1 : 2000); rabbit anti-Akt and p-Akt (1 : 2000). GAPDH (1 : 4000) and β-actin (1 : 4000) were used as internal proteins. The membrane was washed with TBST three times, after that incubated with anti-rabbit or anti-mouse immunoglobulin G horseradish peroxidase-linked secondary antibody (1 : 5000) for 2 h at room temperature. The blotted bands of protein were detected by using chemiluminescence detection kit (Amersham, Shanghai, China). Optical density measurements were obtained by using image analysis software (Bio-Rad Laboratory, U.S.A.).
Enzyme-Linked Immunosorbent Assay (ELISA)Brain tissues were homogenized in ice-cold PBS with mixed protease inhibitor and phosphatase inhibitors. The oxidative stress and inflammation status of the brains were evaluated by measuring catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), malondialdehyde (MDA), interleuin-1β (IL-1β), interleuin-6 (IL-6) and tumor-necrosis-factor-α (TNF-α) kits. The measurement processes were performed according to the manufacture’s instruction using commercial kits (Nanjing Jiancheng Bioengineering Institute).
Statistical AnalysisAll experimental data expressed as mean ± standard error of the mean (S.E.M) were analyzed by SPSS statistical software 16.0. All data were analyzed using one-way ANOVA, Dunnett’s multiple range tests and Student–Newman–Keuls’ test. A value of p < 0.05 indicated a significant difference.
The product (C27H41NO4) was obtained as a light-yellow syrup (1.3 g yield = 50%) (Fig. 1).
A: 13C-NMR spectrum, B: 1H-NMR spectrum, C: mass spectrometry.
1H-NMR (400 MHz, DMSO-d6) δ: 12.53 (s, 1H, –COOH), 9.19 (s, 1H, –PhOH), 8.02 (d, 1H, J = 8.1 Hz, –NH–), 7.11–6.91 (m, 2H of Ph), 6.74–6.55 (m, 2H of Ph), 5.42–5.24 (m, 4H, –CH=CH–CH2–CH=CH–), 4.50–4.14 (m, 1H, –CH–), 2.91 (dd, 1H, J = 13.8, 4.8 Hz, PhCH2–), 2.77–2.67 (m, 3H, 1H of PhCH2– and 2H of –CH=CH–CH2–CH=CH–), 2.06–1.96 (m, 6H, –(CH2–CH=CH)2CH and –COCH2–), 1.45–1.34 (m, 2H of –COCH2CH2–), 1.34–1.16 (m, 14H 7 × CH2), 0.85 (t, 3H, J = 6.8 Hz, –CH2); 13C-NMR (100 MHz, DMSO-d6) δ: 173.31, 172.06, 155.83, 129.90, 129.71, 129.69, 127.71, 127.68, 114.84, 53.60, 36.01, 35.04, 30.87, 29.04, 28.71, 28.67, 28.56, 28.49, 26.63, 26.58, 25.19, 25.17, 21.95, 13.89; electrospray ionization (ESI)-MS: [M + H]+ m/z 443.3036 .
THe Effect of NITyr on Autonomic Activity in the OFTThe total movement distance of gerbil was assessed as autonomous activities and the time spent in the center, corner and side was used to evaluate the exploratory and anxiety-related behavior in the OFT. In Fig. 2, the total movement distance and the time spent in each corner of the field in the ischemia and NITyr group did not show any changes compared with those of the sham group.
(A) The total movement distance of gerbil in the OFT was evaluated as spontaneous locomotor activity. (B–D) the time spent in the center, corner and side were evaluated as anxiety-related behavior of mice. Values are reported as the mean ± S.E.M.
As shown in Fig. 3A, all gerbils exhibited improved motor performances during the training time. On the third day, the time stayed on the rod of the gerbil in the sham group was longer than that of the ischemia group (p < 0.05). On the fifth day, 5 and 10 mg/kg NITyr could reverse the above effects (p < 0.05).
(A) The time on the rotary bar of the gerbil. (B) The escape latency of the gerbil. (C) The swimming time in the target quadrant of the gerbil. (D) The swimming track of the gerbil. Values are reported as the mean ± S.E.M. * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. the ischemia group.
To examine whether NITyr played a role on ischemia-induced memory impairments of gerbil, the escape latency in the MWM was assessed (Fig. 3B). The escape latency of the ischemia group was longer than that of the sham group (p < 0.05, p < 0.01). On the fourth day, only 10 mg/kg NITyr could reverse these effects (p < 0.05). As the extension of training time, 5 and 10 mg/kg NITyr reversed these effects on the fifth day (p < 0.05, p < 0.01). The retention time in the target quadrant was used to assess the learning and memory ability of gerbil by removing the underwater platform after five days of training (Fig. 3C). The exploratory time in the target quadrant of the sham group spent more time than that of the ischemia group. NITyr (5 and 10 mg/kg) reversed the above phenomenon (p < 0.05, p < 0.01). The swimming trajectory of the ischemia group was inappropriate and the NITyr group deviated from the target quadrant for a relatively long time (Fig. 3D). 10 mg/kg NITyr was chose for the following researches.
Effect of NITyr on Histological ChangesHistologic features revealed the characteristics of delayed neuronal death by HE staining and Nissl staining. The microphotography of each group in the CA1 region were shown in Fig. 4. The neurons in the sham group were distinct, medium size and normal ultrastructure. In the ischemia group, the neurons were irregularly arranged, blurred cell outlines, atrophic cell bodies, vacuolization and pyknotic nuclei, which revealed obvious neuronal damage. NITyr (5 and 10 mg/kg) could ameliorate the above phenomenon, reduce neuronal loss and alleviate other morphological changes (p < 0.05, p < 0.01).
Scale bars represented 20 µm. The neurons in the hippocampus were stained with HE staining (A) and Nissl staining (B) (400×). The healthy neuron present clear and evident nuclei and nucleoli. There was significant neuronal damage in ischemia group compared with the sham group. NITyr could reverse the above phenomena.
Neuronal apoptosis is accompanied by inflammation and oxidative stress. In Figs. 5A–C, the levels of TNF-α, IL-6 and IL-1β in the ischemia group were remarkably higher than those of the sham group (p < 0.01, p < 0.001). 10 mg/kg NITyr reversed the above phenomena (p < 0.05, p < 0.01) and 5 mg/kg NITyr only decreased the levels of TNF-α and IL-1β (p < 0.05). Compared to the sham group, the content of MDA in the ischemia group increased significantly, while the activities of SOD and CAT decreased, whereas 10 mg/kg NITyr reversed the above phenomena except GPx (p < 0.05) and 5 mg/kg NITyr only increased the activities of CAT (Figs. 5D–G).
The levels of IL-1β (A), IL-6 (B) and TNF-α (C) in the ischemia group were significantly higher than that of the sham group. In the ischemia group, the activities of CAT (E) and SOD (F) showed a marked decrease and the content of MDA (D) significantly increased compared with the sham group and NITyr reversed the above phenomena. Values are reported as the mean ± S.E.M. * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. the ischemia group.
As shown in Fig. 6, on the fifth day, AM251 and AM630 alone had no effects on animal behaviors and histological changes compared with the ischemia group. Of note, AM630 but not AM251 reversed the effects of NITyr on the time stayed on the rod, the escape latency, the retention time in the target quadrant and the histological changes of the gerbil (p < 0.05, p < 0.01).
(A) The time stayed on the rod of the mice in the rotary test. (B) The escape latency of the mice in the MWM. (C) The retention time in the target quadrant of the mice in the MWM. (D) The histological changes of the gerbil via H&E staining. (E) Numerical analysis of HE staining. (F) The histological changes of the gerbil via Nissl staining. (G) Numerical analysis of Nissl staining. Values are reported as the mean ± S.E.M. * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. the ischemia group; # p < 0.05, ## p < 0.01 vs. the NITyr group.
As shown in Fig. 7, AM251 and AM630 alone had no effects on oxidative stress and inflammation-related factors compared with the ischemia group. AM630 but not AM251 reversed the effects of NITyr on the levels of IL-1β, IL-6 and TNF-α of the gerbil (p < 0.05). Interestingly, AM251 but not AM630 reversed the effects of NITyr on the level of MDA of the gerbil (p < 0.05). Both AM630 and AM251 combined with NITyr have no effect on the activities of SOD and CAT compared with the NITyr group.
IL-1β (A), IL-6 (B), TNF-α (C), MDA (D), CAT (E) and SOD (F) in the brains of the mice were measured using ELISA. Values are reported as the mean ± S.E.M. * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. the ischemia group; # p < 0.05 vs. the NITyr group.
Compared with those of the sham group, the expression levels of CB1 and CB2 decreased in the ischemia group. Whereas NITyr could obviously increase the expression levels of CB2 but not CB1 compared with the ischemia group (Figs. 8A, B). The expression levels of p-PI3K and p-Akt were similar to that of CB2 (Figs. 8C–F). Compared with those of the sham group, the expressions levels of p-PI3K and p-Akt decreased in the ischemia group, whereas these levels increased in the NITyr group. AM630 reversed the effects of NITyr on the expression levels of p-PI3K and p-Akt in the brains of the gerbil but not AM251.
Values are reported as the mean ± S.E.M. * p < 0.05 and ** p < 0.01 vs. the ischemia group; # p < 0.05 vs. the NITyr group.
The incidence of transient global cerebral ischemia is raising in modern times, however few of efficacious drugs are availability to treat it so far.14) The discovery and appliance of a new chemical compound capable of interfering with multiply pathological mechanisms is a novel strategy for ischemia.15) In our previous researches, N-stearoylamino acids as an AEA analogue exerted potent neuroprotective effects and N-stearoyl-tyrosine showed good activity among them.11) However, N-stearoyl-amino acid need to be used at several orders of magnitude higher than AEA to achieve better neuroprotective activity. It has been documented that the compounds with double bonds appear to have more close relationships with AEA.11) Considering linoleic acid with two double bonds has high stability and the activities of N-stearoyl-tyrosine was benign, we have synthesized NITyr and evaluate its pharmacological activity.
The gerbils could be used to induce global ischemia in the study because the developed features of global cerebral ischemia were prone to occur in gerbils only by temporary occlusion of common carotid arteries, which could produce more severe forebrain damage.16) It is believed that ischemic injury in the hippocampus lead to serious damages of spatial memory and neuronal loss. To better understand the effects of NITyr on global ischemia, both the cognitive and non-cognitive behaviors of the gerbil were evaluated, for example locomotion, exploration, and anxiety-related behavior (OFT), motor function (RRT) and the ability of learning and memory (MWM).17,18) NITyr had no effects on the autonomic activity, investigating behavior and anxiety behavior in the OFT, but increased the motor function in the RRT during the extension of the training time, indicating that NITyr could improve the motor function of the gerbil through enhancing the ability of learning and memory, but not affecting motor function. The improvement of learning and memory impairment might be related to the repairmen of damaged brain tissue rather than the improvement of motor ability of the gerbil.19) These results indicated that NITyr did not affect the non-cognitive behaviors of the gerbil, but specifically affected the cognitive behaviors. Meanwhile, NITyr could improve the learning disorders at 5 and 10 mg/kg, there was no memory retention in NITyr group at 1 mg/kg, indicating that NITyr exhibited good activity in a certain dose. Thus, we selected 10 mg/kg NITyr for subsequent experiments. Nimodipine has been widely used in the treatment of brain injury,20) there it is used as a positive drug in the experiment.
The pronounced deletion of the neurons in the hippocampus is related to aggravated damages of animal behavior disorder, such as the performance in spatial learning and memory.21) The present studies confirmed that more than 80% of the neurons in the hippocampus were damaged after a 5-min occlude in HE staining and Nissl staining. It was also noticeable that NITyr significantly reduce neuronal damage and provided adequate protection for the hippocampus, including neuronal loss, smaller size, irregular shape, deepened cytoplasm, and atrophic nuclei. The observed diminution of neuronal loss after the treatment of NITyr was consistent with the results of MWM.
The neuronal death in the hippocampus is accompanied by inflammatory response and oxidative stress, which could eventually induce apoptosis.22) Accordingly, NITyr could obviously decrease the release of inflammatory cytokines that could induce the inflammatory cascade, thereby ameliorate central nervous systems disorders.23) Meanwhile, lipid peroxidation products and the activities of antioxidant enzymes were strikingly ameliorated in the NITyr group consisted with our previous observations of N-stearoyl amino acids in ischemia-reperfusion injury.6) NITyr could reduce the level of MDA and enhance the activities of SOD and CAT but not GPx. The possible results might be that NITyr could inhibit the activation of microglia that could produce reactive oxygen species. Meanwhile, 5 mg/kg NITyr only ameliorated the activities of CAT but not SOD, indicating that NITyr exhibited good activity in a certain dose.
Both CB1 receptor and CB2 receptor were expressed in the brain of the gerbil. We found that NITyr-induced the amelioration of the animal behavior could be fully reversed by CB2 receptor antagonist AM630 but not AM251. These results indicated that NITyr ameliorated the animal behaviors via CB2 pathway. Moreover, the effects of NITyr on inflammation could be reversed by AM630 but not AM251. However, the effects of NITyr on oxidative stress could be reversed by AM251 but not AM630. The above phenomena might be because CB2 receptor are more likely to be distributed in the immune system, which are closely related to inflammation.24) These results indicated that NITyr could exert different biological effects through different receptors, among which CB2 receptor play a greater role than CB1 receptor.
The CB1 and CB2 signaling mechanism is associated with the activation of the PI3K/Akt pathway.25,26) The PI3K/Akt pathway exhibit neuroprotection against brain injury that is associated with animal behavior.27,28) Furthermore, NITyr could activate PI3K pathway, which could be reversed by CB2 receptor antagonists rather than CB1 receptor antagonists. Meanwhile, NITyr could upregulate the expression of CB2 but not CB1. These results indicated that NITyr could activate PI3K pathway through CB2 receptor but not CB1 receptor.
A number of studies showed that AEA might play key neuroprotective effects in either acute neuronal injury or various chronic neurodegenerative disorders via CB2 receptor to regulate the main pathological processes, such as excitotoxicity, inflammation and oxidative stress etc.29) The present study firstly demonstrated that NITyr as AEA analogue could ameliorate animal behaviors in cerebral ischemia in gerbils, increase cell viability in the hippocampus, and improve inflammation and oxidative stress via CB2 receptor involvement of PI3K/Akt pathway; it might become a good candidate for treating cerebral ischemia.
This work was financially supported by the Natural Science Foundation of China (No.81803514), Sichuan Science and Technology Innovation Seedling Project (No. 2018011), the National Student Innovation Training Program (Nos. 508-2033027, 508-2033055), and the Fund Project of Sichuan Provincial Department of Education (No.18ZB0165).
The authors declare no conflict of interest.
