Meloxicam improves cognitive impairment of diabetic rats through

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Meloxicam improves cognitive impairment of diabetic rats through COX2-PGE2-EPs-cAMP/pPKA pathway Huan Li, Ying Luo, Ying Xu, Lu Yang, Congli Hu, Qi Chen, Yang Yang, Jie Ma, Jiahua Zhang, Hui Xia, Yuke Li, and Junqing Yang Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00532 • Publication Date (Web): 15 Aug 2018 Downloaded from http://pubs.acs.org on August 16, 2018

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Molecular Pharmaceutics

Meloxicam improves cognitive impairment of diabetic rats through COX2-PGE2-EPs-cAMP/pPKA pathway Huan Li1, Ying Luo1, Ying Xu2, Lu Yang1, Congli Hu1, Qi Chen1, Yang Yang1, Jie Ma1, Jiahua Zhang1, Hui Xia1, Yuke Li1 and Junqing Yang1* 1 the Key Laboratory of Biochemistry and Molecular Pharmacology, Department of Pharmacology, Chongqing Medical University, Chongqing 400016, China. 2 Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, State University of New York at Buffalo, NY 14214, USA. *

Correspondence: Prof. Junqing Yang. E-mail: [email protected],

[email protected]; Tel: +86-23-68485161; Fax: +86-23-68485161 Co-first authors: Huan Li, Ying Luo and Ying Xu Abstract Diabetics often face greater risk of cognitive impairment than non-diabetics. However, how to prevent this disease is still unconfirmed. In this study, we investigated the potential protection and mechanism of meloxicam on cognitive impairment in diabetic rats. Diabetic rat model was established with high-fat diet and a small dose of streptozotocin (40 mg/kg). The changes of spatial learning and memory, histopathology and the protein expressions of amyloid protein precursor (APP) and β-amyloid (Aβ) indicated that diabetic rats had neuronal injury and cognitive impairment. Tumor necrosis factor α (TNFα), interleukin 6 (IL-6), C reactive protein (CRP) and prostaglandin E2 (PGE2) levels and microglial cell number were significantly increased in the diabetic rat brain. Meanwhile, the protein expressions of APP, Aβ, cyclooxygenases2 (COX2), E-type prostanoid recptors 1 (EP1) and EP2, and the level of cyclic adenosine monophosphate (cAMP) were significantly increased,

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while the protein expressions of EP3 and phosphorylated protein kinase A (pPKA) were significantly decreased in the diabetic rat hippocampus and cortex. However, the EP4 protein expression had no significant changes. Meloxicam significantly improved neuronal injury and cognitive impairment, and significantly decreased inflammatory cytokines levels. Meloxicam also significantly decreased the protein expressions of APP, Aβ, COX2, EP1 and EP2 and the level of cAMP, and significantly increased the EP3 and pPKA protein expressions in rat hippocampus and cortex. However, meloxicam did not significantly influence the levels of blood glucose, lipids and insulin of rats. Our results suggest that meloxicam could significantly protect diabetic rats from cognitive impairment via a mechanism that may be associated with rebalancing the COX2-PGE2-EPs-cAMP/PKA pathway. Keywords: diabetes, cognitive impairment, inflammation, cyclooxygenase 2, meloxicam Introduction In the world today, diabetes has become one of the most challenging diseases. The number of cases of diabetes has exceeded 300 million worldwide and is increasing year by year.1-2 Diabetes has serious effects on the quality of patients' life and is associated with many novel complications, such as diabetic retinopathy, diabetic nephropathy and diabetic neuropathy,3 which affect nearly 30% of diabetics and have become a focus of research.4 Patients with diabetic neuropathy often suffer from cognitive impairment characterized by lower learning, memory and language abilities and have a higher risk progressing to Alzheimer’s disease (AD) than the general population.5-8 Although the mechanism of diabetic cognitive impairment is still unconfirmed, some studies have shown that it is associated with inflammation.9-11 One of foremost pharmacological functions of non-steroidal anti-inflammatory drugs (NSAIDs) is anti-inflammation, the mechanism of which is related to inhibition of cyclooxygenase 2 (COX2). Meloxicam has been shown to improve scopolamine-induced cognitive


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impairment and object recognition memory after splenectomy in mice.12-13 Liu et al. proved ibuprofen could improve diabetic encephalopathy by down-regulating β-amyloid precursor protein cleaving enzyme 1 (BACE1), the the advanced glycation endproducts/receptors of advanced glycation endproducts (AGEs/RAGE) axis and inflammation mediators in diabetic rat brain.61 Both meloxicam and ibuprofen play a major pharmacological role in inhibiting COX2, however, there are few reports about their inhibition of COX2 to improve cognitive impairment in diabetes. Therefore, we focused on COX2 and its related pathways. Cyclooxygenase (COX), consisting of structural COX1 and inducible COX2 subunits, is the rate-limiting enzyme of prostaglandin (PG) biosynthesis. COX1 is expressed at high levels and plays a crucial physiological role in most cells and tissues, such as in vascular endothelial cells, platelets, renal collecting tubules, stomach and smooth muscle.14 COX2, which is expressed at low or undetectable levels in some normal tissues but at high levels in inflammatory states, is important in many organs and systems, including kidney, brain, bones and so on.14 COX2 is expressed in many brain diseases, such as global ischemia, AD and depression. Furthermore, COX2 inhibitors have a protective effect on brain damage.15-17 Clinical researches have confirmed that long-term use of COX inhibitors can reduce the prevalence of AD by affecting inflammation, neuronal loss and behavioral changes.18-19 However, long-term use of non-selective COX inhibitors can cause adverse reactions in the gastrointestinal, liver, nervous and cardiovascular systems and other adverse reactions, possibly by simultaneous inhibition of both COX1 and COX2. It has been reported that meloxicam, a selective COX2 inhibitor, is associated with a significant reduction in adverse reactions.20 Among the PGs produced by COX-mediated oxidation of arachidonic acid, prostaglandin E2 (PGE2) is not only the most plentiful prostanoid in humans, but also plays an important part in various physiological functions and pathophysiological processes.21 PGE2 participates in cerebral inflammation and the level of it is significantly increased in brain injury.22-23 Some studies have suggested that reducing ACS Paragon Plus Environment


PGE2 level could have a protective effect on brain injury.24-25 Our previous study showed that the four different receptor subtypes of PGE2 participated in brain injury caused by aluminum overload.26 Based on these reports, we hypothesized that COX2 and its downstream signaling pathway may participate in cognitive impairment in diabetic rats and that a COX2 inhibitor will have a protective effect on diabetic cognitive impairment. In this study, we investigated the protection of meloxicam on cognitive impairment in diabetic rats as well as the alterations of COX2, PGE2 and its downstream pathway. Materials and Methods Animals Sixty male Sprague–Dawley rats (aged 4 weeks, weight 180–200 g) were purchased and raised at the Laboratory Animal Center, Chongqing Medical University, China (License number: SYXK YU 2012-0001). All experiments were permitted by the Laboratory Animal Administrative Center and the Institutional Ethics committee of Chongqing Medical University and were conducted according to the National Institutes of Health guide for the laboratory animals. All applicable institutional and national guidelines for animals were followed. All rats were maintained on a standard laboratory diet. Meloxicam preparation 10.8 mg meloxicam (Solarbio, China) was weighed and fully ground, and then 40 ml 0.5% CMC-Na was added into it to prepared into 0.27 mg/ml meloxicam suspension. 10 ml of meloxicam suspension (0.27 mg/ml) was measured, then was added into 20 ml of 0.5% CMC-Na to prepare the 0.09 mg/ml of meloxicam suspension. Diabetes model The diabetic rat model was prepared as previously described.27 After being fed in the experimental environment for seven days to acclimatize, rats were then divided into


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normal diet (ND) group (n=10) and high-fat diet (HFD) group (n=50). The ND group (n=10) was fed with a standard laboratory diet and the HFD group was fed with a formulation comprising 20% sucrose, 10% lard, 10% yolk powder and 60% basal fodder, for 4 weeks to induce insulin resistance. All the rats were then fasted for 12 h and the HFD group received a peritoneal injection of streptozotocin (STZ, Solarbio, China) at a dose of 40 mg/kg to establish the diabetes model, while the normal group (n=10) received the same volume of vehicle (citrate buffer solution). Diabetic rats were identified as those with fasting glucose higher than 16.7 mmol/L after 72 h. The diabetic rats were continuously fed with a HFD for 4 weeks and then randomly divided into a diabetic model group, a meloxicam (0.9 mg/kg) group and a meloxicam (2.7 mg/kg) group (n = 10 rats per group). The dose of meloxicam was based on our previous researches, researches by other researchers and our pre-experimental results.39,62-65 Rats in the meloxicam groups were administered the drug by gastric perfusion, while rats in the normal group and the model group were given the same amount of vehicle (0.5% CMC-Na), all drugs were given for 8 weeks, once a day. Body weight and blood biochemistry measurement From week 9, blood glucose and body weight were measured every three weeks. Five rats from each group were used to measured plasma insulin levels by enzyme-linked immunosorbent assay (ELISA, Yuanye, China). Triglycerides (TG), total cholesterol (TC) and low density lipoprotein cholesterol (LDL-C) levels were also tested by chemical kits (Nanjing, China). All operations were carried out in accordance with the instructions. Morris water maze test At week 18, Morris water maze test was performed in all rats as previously described.28 The temperature of the water was maintained between 20°C and 25°C. The test program included two stages, with the first stage was the navigation training, which lasted for 4 days. The rats were put into the water randomly from four positions with the head of the animal facing toward the wall. The time taken for the rats to find the



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platform (escape latency) was recorded. When the time exceeded 90 s, the animal was guided to the platform and allowed to rest on it for 10 s. The second stage was the space detection, which was tested on day 5. The platform was removed and each rat was put into the water from the last training position, the escape latency (with a maximum of 90 s) and the frequency that the rats crossing the platform were recorded. Histopathological observations When the Morris water maze test was over, rats (n = 3 per group) were sacrificed followed by perfusion with phosphate buffered saline (PBS). The brains of the rats were removed and fixed in 4% paraformaldehyde (pH 7.2) before being embedded in paraffin. Sections of the brain (thickness, 5 µm) were prepared and stained with hematoxylin-eosin (HE). Morphologic alterations of the cortex and hippocampal nerve cells were observed by microscopy. All zones of the hippocampus were observed and the damaged subarea was taken. Immunohistochemistry Immunohistochemical staining was performed to test the expression of ionized calcium-binding

adapter

molecule

1

(IBA1),

the

marker

of

microglia.

Paraffin-embedded sections of the brains of three rats from each group were dewaxed with xylene (two washes for 10 min) and rehydrated with graded ethanol (100%, 95%, 80% and 60%; 5 min per wash). Then 3% hydrogen peroxide were dropped onto the sections to eliminate endogenous peroxidase activity. After 10 min, The sections were rinsed with distilled water and immersed in PBS for 5 minutes. Antigen retrieval was performed with citrate buffer in the microwave on medium power for 10 min and the sections were incubated with sheep serum (1:10 dilution, ZSGB-BIO, Beijing) at room temperature for 15 min. The sections were then incubated with a primary antibody for the detection of IBA1 (1:20, Proteintech, China) overnight at 4°C. After rewarming at room temperature for 30 min, the sections were rinsed with PBS (3 times,3 min per wash). Subsequently, the sections were covered with secondary antibody (1:300, ZSGB-BIO, Beijing) for 30 min at room temperature and rinsed with PBS (3 times, 3


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min per wash), then incubated with streptavidin labelled with horseradish peroxidase for 30 min at room temperature. After being rinsed with PBS, the sections were dyed with 3, 3- diaminobenzidine (DAB) for 3 s and washed by tap water, then counterstained with hematoxylin. Finally, the sections were mounted and observed under a light microscope. Enzyme-linked immunosorbent assay After sacrifice, the hippocampus and cortex of the rats (n = 4 per group) were placed on ice immediately and homogenates were prepared with normal saline (w/v, 1:9). The supernatants were collected for the analysis of PGE2, cyclic adenosine monophosphate (cAMP), interleukin 6 (IL-6), tumor necrosis factor α (TNFα) and C reactive protein (CRP) levels using ELISA kits (Yuanye, China) according to the instructions. Western blot analysis Proteins were extracted from the hippocampus and cortex of rats (n =3 per group) using tissue lysate reagent (Dingguo, China), the concentrations of which were measured with BCA protein assay kits (Beyotime, China) according to the instructions. Protein samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. Then membranes were covered with 5% bovine serum albumin at room temperature for 2 h and incubated with primary antibodies overnight at 4°C for detection of COX-2 (1:1,000, Abcam, UK), amyloid protein precursor (APP, 1:500, Boster, China), β-amyloid (Aβ, 1:1,000, Abcam, UK), E-type prostanoid recptors 1 (EP1, 1:400, Boster, China), EP2 (1:1,000, Abcam, UK), EP3 (1:400, Bioss, China ), EP4 (1:400, Proteintech, China), phosphorylated protein kinase A (pPKA; 1:1000, Abcam, UK) and β-actin (1:1,000, Proteintech, China). After being washed with Tris Buffered Saline with Tween 20 (4 times, 5 min per wash), the membranes were incubated with secondary antibodies (1:1,000, Proteintech, China) at room temperature for 1 h. Finally, protein bands were detected with Bio-Rad imaging system (USA).



Statistical analysis Data were reported as mean ± SD. All analyses were performed using SPSS 17.0. Data variation was evaluated with one-way analysis of variance (ANOVA) followed by Dunnett’s t-test. P < 0.05 was indicated statistical significance. Results Changes in blood glucose, body weight, plasma insulin levels and blood biochemistry in diabetic rats In the experiment, 12 rats were eliminated for modeling failure, 8 rats died during the period of model establishment, and another 8 rats died during the period of drug administration (3 in the model group, 3 in the meloxicam (0.9 mg/kg) group, and 2 in the meloxicam (2.7 mg/kg) group). Autopsies did not reveal any obvious abnormalities in the peripheral tissues and organs. Fasting blood glucose of the rats was tested every three weeks. Fasting blood glucose in the diabetes model group was significant increased (P

Meloxicam improves cognitive impairment of diabetic rats through

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