Ethanol extract of Alpiniae oxyphyllae fructus regulates glucose metabolism, the HPA axis and hippocampal function in diabetic mice with depression

doi:10.21203/rs.3.rs-3545438/v1

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Ethanol extract of Alpiniae oxyphyllae fructus regulates glucose metabolism, the HPA axis and hippocampal function in diabetic mice with depression

https://doi.org/10.21203/rs.3.rs-3545438/v1

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Background: Alpiniae oxyphyllae fructus (AOF) is the dry ripe fruit of Alpinia oxyphylla Miq. which has significant therapeutic effects in Alzheimer's disease, Parkinson's disease, depression, learning and memory dysfunction and other nervous system disease.

Methods: In this study, we established a mouse model of Type 2 Diabetes Mellitus (T2DM) combined with depression induced by feeding high fat and high sugar diet combined with intraperitoneal injection of streptozotocin (STZ) and chronic unpredictable mild stress (CUMS) to evaluate the dual therapeutic effects of ethanol extract of AOF (EEA), and further explore the mechanism of EEA on diabetes complicated with depression.

Results: The results showed that EEA improved the body weight of diabetic mice with depression (DD mice). EEA could also improve glucose metabolism and insulin resistance in DD mice. Next, EEA improved the depression-like behaviors of DD mice. EEA also attenuated diabetes and CUMS-induced hyperactivity of the hypothalamic-pituitary-adrenal(HPA) axis and improved the expression of neurotransmitters and synaptic plasticity in the hippocampus of DD mice. In addition, EEA could improve hippocampal tissue damage caused by neuroinflammation and oxidative stress. Further research showed that EEA improved the protein expression and phosphorylation of Phosphoinositide 3-kinase (PI3K), Protein kinase B (Akt) and Mammalian target of rapamycin (mTOR) in the hippocampus of DD mice.

Conclusions: This study showed that EEA may have a dual role in the treatment of diabetes and depression and provided new scientific theoretical basis for the future development of AOF and the prevention and treatment of diabetes complicated with depression.

Diabetes

CUMS

Insulin resistance

Neuroinflammation

Synaptic plasticity

Neurotransmitter

Type 2 diabetes mellitus (T2DM) is a systemic metabolic disease characterized by chronic hyperglycemia^[1]. According to the International Diabetes Federation (IDF), as for 2021, there were more than 537 million diabetes patients in the world, accounting for about 10.5% of the global population^[2]. T2DM not only has a serious harm to human health, but its complications will also bring a heavy burden on family and society^[3]. As a chronic metabolic disease, T2DM may lead to complications of cardiovascular system, peripheral vascular system, nervous system and other systems with the development of the course of the disease^[4]. Depression is one of the most prevalent mental health disorders that limits psychosocial functioning, reduces quality of life, and is a common psychological complication of diabetes^[5]. The results of a recent prospective study also showed a higher prevalence of depressive disorders in patients with diabetes than in the general population^[6]. An existing study has shown that patients with diabetes are generally faced with psychological disorders such as anxiety and depression, which lead to adverse clinical outcomes, such as reduced quality of life, increased treatment costs, and increased complications and mortality^[7].

The disease progression rate of diabetic patients with depression is faster and the mortality rate is also increased, which bring great difficulties to drug treatment^[8]. Treatments for diabetes with depression include conventional hypoglycemic and antidepressant therapies. However, the presence of depressive symptoms can lead to poor medication adherence, poor lifestyle, and unhealthy eating habits in diabetes patients with depression, which in turn affects the therapeutic efficacy of medications^[9]. In addition, the current drug options for treating diabetes with depression are few and may induce significant side effects^[10]. For example, a cohort study showed that antidepressant use is associated with the risk of diabetes onset in a time- and dose-dependent manner, and could induce blood-sugar related disorders^[11]. The results of a systematic review and meta-analysis suggest that the use of antidepressants in patients with major depressive disorder can lead to gastrointestinal side effects^[12]. Therefore, it is urgent to explore the pathogenesis of diabetes and develop drugs that can improve both diabetes and depression.

Traditional Chinese Medicine (TCM) is one of the oldest treatment systems^[13]. Due to the inherent advantages of TCM with multiple targets and multiple components, its traditional application in modern medicine has been expanding^[14]. At present, the efficacy of traditional Chinese medicine in the treatment of diabetes combined with depression has been widely studied^[15–17]. Alpiniae oxyphyllae fructus (AOF) is the dry ripe fruit of Alpinia oxyphylla Miq. which is widely used in diarrhea, vomiting, frequent urination, and so on in TCM^[18]. In addition, more effects of AOF have been reported, such as neuroprotection, improvement of learning and memory ability, anti-inflammatory, antioxidant, anti-aging and other functions^[19]. An existing study found that AOF can reduce blood glucose levels in diabetic mice, and also has a better effect on diabetic complications such as diabetic nephropathy^[20]. A large number of studies have confirmed that AOF has significant therapeutic effects in Alzheimer's disease, Parkinson's disease, depression, learning and memory dysfunction and other nervous system diseases^[21–24]. Among them, the salutary effect of AOF on depression has been widely studied. Studies have shown that AOF can improve depressive behavior and neuroinflammation in mice exposed to Chronic Unpredictable Mild Stress (CUMS)^{[23, 25]}. However, whether AOF has dual effects on the treatment of diabetes and depression still needs further exploration.

In this study, we established a mouse model of T2DM combined with depression induced by feeding high fat and high sugar diet combined with intraperitoneal injection of streptozotocin (STZ) and CUMS to evaluate the dual therapeutic effects of AOF, and further explore the mechanism of AOF on diabetes complicated with depression.

2.1 Chemicals

Metformin was produced by Bristol-Myers Squibb, USA. Fluoxetine was purchased from Eli Lilly, USA. Streptozocin (STZ) was purchased from Sigma-Aldrich, USA. The biochemical assay kit for monoamine oxidase (MAO), total antioxidant capacity (T-AOC), malondialdehyde (MDA) and superoxide dismutase (SOD) were purchased from Nanjing Jiancheng Biotechnology Institute (Nanjing, China). The enzyme-linked immunosorbent assay (ELISA) kits for fasting insulin (FINS), glycated hemoglobin (HbA1c), dopamine (DA), 5-hydroxytryptamine (5-HT), norepinephrine (NE), corticotropin-releasing factor (CRF), corticosterone (CORT), adrenocorticotrophic hormone (ACTH), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) were purchased from Wuhan Huamei BIOTECH Co., Ltd. (Wuhan, China). The main antibodies and secondary antibodies were purchased from Wuhan Sanying Biology Technology Co., Ltd (Wuhan, China). The anti-phospho-antibodies were purchased from Cell Signaling Technology Company (CST, USA).

2.2 Plant materials and preparation of ethanol extract of AOF (EEA)

Dried AOF were purchased from Zhang Zhongjing Pharmacy (Zhengzhou, China). AOF were washed 3 times with pure water, and then placed in an electric air blast drying oven (DHG-9076A, Shanghai, China) at 45℃ to dry the water to a constant weight. Then, the dried AOF was ground into powder in a high-speed grinder (XFB-200, Hunan, China), passed through a 40 mesh screen, and stored in a refrigerator at 4°C in the dark.

The AOF powders were degreased with carbon tetrachloride (w/v = 1 : 10 g mL^− 1) for 24 h, filtered and dried in the fume hood to obtain the defatted dry powder of AOF. The defatted powders were extracted with 70% ethanol solution (w/v = 1 : 10 g mL^− 1) at room temperature for 24 h in the dark then the supernatant was collected, repeat the extraction for two more times. The filtrates obtained from three-time extracted defatted powders were combined and concentrated using a rotary evaporator (YRE2000B, Henan, China). Then the concentrated solutions were frozen at − 80°C in a refrigerator (DW-86L386, Shandong, China) and freeze-dried in a vacuum freeze dryer (LGJ-18B, Beijing, China). The freeze-dried powder was stored at 4°C until further use.

2.3 Animals

A total of 48 male specific-pathogen-free C57BL/6J mice (18gཞ20 g, 6 weeks) were purchased from Beijing SBF Biotechnology Co., Ltd (Beijing, China). Mice were housed under conditions of no specific pathogens at an ambient temperature of 22°C to 25°C, humidity of 50 ± 5%, and a 12/12-h light-dark cycle with food and water. All animal procedures were performed in accordance with the National Research Council's Guide for the Care and Use of Laboratory Animals.

2.4 Experimental design

After one week of acclimatization, the mice were randomly divided into normal control group (NC group) (n = 8) and model group (n = 40). The normal control group was fed with a normal pellet diet (5.2% fat, 61.0% carbohydrate, and 18.8% protein, total energy: 3660 kcal kg^− 1) while the model group was fed with a high-fat diet (34.55% fat, 27.20% carbohydrate, and 23.25% protein, total energy: 5128 kcal kg^− 1). In week 8, all mice were fasted for 12 h; the mice in the normal group were injected with citrate buffer, and the model group mice were injected with STZ at 150 mg kg^− 1 for two consecutive days. The levels of Fasting blood glucose (FBG) at 72 h and 1 week were measured. When the FBG level is ≥ 11.1 mmol/L for two consecutive times, it meets the criteria of diabetes model. The diabetes combined with depression was established by CUMS combined with solitary confinement in mice conforming to the diabetic model. The sucrose preference test and forced swimming test were performed before and after the establishment of diabetes combined with depression model.

The diabetic mice with depression (DD mice) were randomly divided into five groups (n = 8 mice in each group) namely model comparison group (DD group), positive control group (PC group), EEA low-dose group (EEA-L group), EEA medium-dose group (EEA-M group) and EEA high-dose group (EEA-H group). The positive control group was given gavage of metformin at a dose of 150 mg kg^− 1 and fluoxetine at a dose of 3 mg kg^− 1 every day, the EEA low-dose group, the EEA medium-dose group, and the EEA high-dose group were given 150 mg kg^− 1, 300 mg kg^− 1, and 450 mg kg^− 1 EEA sample. The control group and the model comparison group were given the same amount of normal saline by gavage for four weeks.

The CUMS protocol was performed as described previously but with a slight modification^[26]. The mice in the model group were administered the following mild stressors randomly for 28 days: swimming in 4°C cold water for 5 min, shocked 8 times with 0.5 mA current, 15 s apart for 20 s, tail pinch for 1 min, shaking at shaker for 15 min, food deprivation for 24 h, tilted cage for 24 h, tail suspension for 5 min and restraint for 2 h. During the study, body weight and fasting blood glucose (FBG) were measured. After the last treatment, the depression-like behaviors in mice were evaluated using the open-field, sucrose preference and forced swimming tests.

The mice were anesthetized and sacrificed on the second day after the behavioral tests. The blood sample was collected from each mouse and centrifuged at 3500 rpm for 10 min. The brain was placed in a Petri dish on ice, and the hippocampus was quickly removed. Part of the hippocampus was fixed in 4% paraformaldehyde solution, and the other part was moved to -80°C for frozen storage. Figure 1 showed the experimental process.

2.5 Oral glucose tolerance test (OGTT)

The OGTT was performed on each mouse on the last day of week 18 to assess glucose tolerance ability of mice from every group. After 12 hours of fasting, all the mice were supplied with 2 g kg^− 1 glucose. The tested blood samples were obtained from the tail vein, and the concentration of blood glucose was determined by glucometers in the next 2 hours, at 0, 30, 60, 90, and 120 min, respectively. The value of the area under the curve (AUC) of blood glucose was calculated^[27].

2.6 Behavioral Tests

2.6.1 Open Field Test (OFT)

In order to evaluate the effects of EEA on the autonomous behavior and exploratory behavior of experimental animals in new and different environments, we used the OFT as previously described for assessment^[28]. The bottom of the observation cage (50 × 50 × 40 cm) was divided into 16 squares of 12.5×12.5 cm. Each mouse was positioned in the middle of the square of the observation cage and allowed to explore freely for 2 min before the test. The behavior of each mouse was recorded in the next 5 min. The following data were recorded: total movement distance and the time of rest. After each animal experiment, the observation cage was cleaned with a 70% ethanol solution to eliminate the influence of the odor left by the previous mouse.

2.6.2 Sucrose preference test (SPT)

The SPT was per-formed using previously reported methods with modifications^[29]. Before testing, mice were supplied with one bottle of 1% sucrose water (w/v) and one bottle of purified water for 24 h. Then, the mice were fasted and deprived of water for 12 h. Pre-weighed sucrose and distilled water bottles were placed on the cages, and the mice drank water freely for 6 h. During this period, the position of the two water bottles was changed every 3 h. At the end of the experiment, the two water bottles were weighed. Sucrose preference (%) = (weight of sucrose consumed/total weight of liquid consumed) × 100%.

2.6.3 Forced swimming test (FST)

The FST was carried out according to published protocols, with minor modifications^[30].The mice were placed in a cylindrical bucket (50 cm height, 20 cm diameter) filled with water (water depth:40 cm, temperature 25°C ± 1°C) for 6 min. The immobility time was recorded from the second minute to the sixth minute by experimenters, and then the mice were dried with a dryer. After each animal experiment, the water in the bucket was replaced to eliminate the influence of the odor left by the previous mouse.

2.7 Biochemical measurements

The levels of FINS, HbA1c, CRF, CORT, and ACTH were determined using an enzyme-linked immunosorbent assay kit. Homeostasis model assessment of insulin resistance (HOMA-IR), used to quantify insulin resistance, was determined based on the following formula: HOMA-IR = FBG (mmol L^− 1) × fasting serum insulin (µ IU mL^− 1)/22.5.

The level of T-AOC and the activities of MDA, SOD and MAO in hippocampus were measured according to the kit instructions. The levels of 5-HT, NE, DA, TNF-α, IL-1β, and IL-6 in hippocampus were determined using an enzyme-linked immunosorbent assay kit. All the experiment procedures followed the instructions of the ELISA kits.

2.8 The histopathology of the hippocampal tissues

2.8.1 Hematoxylin and eosin (H&E) staining

Hippocampus tissue was fixed with 4% paraformaldehyde for 24 hours. The tissues were embedded in paraffin and cut into 4 µm thick slices for pathological and morphological evaluation. The sections were stained with hematoxylin and eosin, and the pathological damage of the hippocampus was observed and analyzed using a microscope equipped with a charged couple device (CCD) camera (BX-51, Olympus, Tokyo, Japan).

2.8.2 Nissl staining

Nissl staining was used to detect the pathological changes in the hippocampus of mice. First, paraffin sections were deparaffinized with xylene and concentration gradient ethanol, and the deparaffinized tissue sections were stained with toluidine blue for 1 h at 50 ° C. After staining, sections were rinsed with distilled water, soaked in 95% ethanol for 5 min, dehydrated by washing with 70%, 80%, 90%, 100% ethanol for 5 min, cleared with xylene for 5 min, and finally sealed with neutral balsam. The pathological damage of hippocampus was observed and analyzed by microscope (BX-51, Olympus, Tokyo, Japan), and the number of Nissl bodies was counted by Image J 1.48 software.

2.9 Immunohistochemistry (IHC)

First, samples were deparaffinized and pre-rinsed with phosphate buffer at 100°C, followed by peroxidase blocking for 5 min. The tissue was washed, and then sheep serum was used to block the nonspecific antigen antibody reaction for 1 h and incubated with the primary antibody Postsynaptic density-95 (PSD-95), Synaptophysin (SYN) and Growth associated protein-43 (GAP-43) (Wuhan Servicebio Technology Co., Ltd.) at 4°C overnight. The next day, after thorough rinsing with PBS, reaction enhancer was added and incubated at room temperature for 30 min. After rinsing with PBS again, DAB chromo developer was added. After color development, the dye was counterstained with hematoxylin staining solution.

2.10 Western blot (WB) analysis

Hippocampal samples were homogenized in ice-cold RIPA lysis buffer containing a mixture of a protease inhibitor and a phosphatase inhibitor and were centrifuged at 12 000 rpm for 10 min in 4°C. After centrifugation, the supernatants were collected and the protein concentration was measured using the BCA kit. After the protein concentration was determined, protein loading buffer was added to the protein samples, which was mixed and placed at 95°C for 5 min to denature the protein, and then cooled at room temperature and stored at -80°C for later use. Aliquots of protein samples were resolved by SDS-PAGE (7.5–15%) and transferred to PVDF membranes. Membranes were blocked with 5% BSA, followed by incubation at 4°C overnight with diluted primary antibodies. Membranes were washed and incubated with secondary antibodies for 1 h at room temperature. Finally, the protein bands were exposed and developed on a chemiluminescence imager, and the results were analyzed using Image J 1.48 software.

2.11 Statistical analysis

IBM SPSS 25.0 was used for statistical analysis of the data. Data were described as mean ± standard deviation (SD). Group differences were determined by using a one-way analysis of variance (One-way ANOVA) with least-significant difference (LSD) test and Dunnett t test. The p value less than 0.05 was considered statistically significant.

3.1 Effects of EEA on body weight and glucose metabolism in DD mice

In our study, for the purpose of evaluating abnormalities in glucose homeostasis in DD mice, the levels of the body weight, FBG, HbA1c and OGTT were measured. In addition, the serum samples were used to examine FINS level and calculated the homeostasis model HOMA-IR index. As can be seen from Fig. 2A, after 4 weeks of drug intervention, the body weight of mice in each intervention group increased compared with that in DD group, and the difference was statistically significant (P < 0.05). Figure 2B shows the blood glucose changes in mice during modeling and intervention. Before the establishment of diabetes model (week 8), the FBG value of mice in each group was at the same level. After STZ injection (week 10), the level of FBG in each intervention group was higher than that in NC group, indicating that the diabetes model was successfully constructed. After 4 weeks of intervention (week 18), the FBG level of all EEA dose groups and PC group decreased compared with DD group, and the difference was statistically significant (P < 0.05).

Insulin resistance is one of the major pathogenesis in T2DM, which can be evaluated by HOMA-IR. As shown in Fig. 2C and Fig. 2D, FINS and HOMA-IR levels in the DD group mice were markedly higher than those in the NC group mice (P < 0.05). Administration of EEA markedly decreased the FINS and HOMA-IR levels compared with that in the DD group mice (P < 0.05). HbA1c reflects stable glucose metabolism in diabetic cases. HbA1c levels were significantly high in DD groups mice after modeling (P < 0.05), compared with the NC group (Fig. 2E). However, EEA treatment significantly reduced HbA1c levels in the DD mice (P < 0.05). This suggested that even though diabetes and CUMS could exacerbate serum HbA1c retention in diabetic mice, EEA treatment could ameliorate this pathophysiology. The mice in the DD group had impaired glucose tolerance and the AUC of their OGTT was higher than that of the NC group (Fig. 3A and Fig. 3B). After the treatment, glucose tolerance levels noticeably ameliorated in the PC group and EEA groups (P < 0.05). These results indicated that EEA may have an effect on maintaining glucose homeostasis in DD mice.

3.2 Effects of EEA on depressive behavior in DD mice

Behavioral tests, including the SPT, FST, and OFT, were conducted to examine the effects of EEA on depression-like behaviors. As shown in Fig. 4A (SPT results), the mice exposed to CUMS for 4 weeks had a significantly decrease in sucrose preference, compared with NC group mice (P < 0.05). After 4 weeks of treatment, DD group mice still had a lower preference for sugar water than the NC group mice, but the preference rate of sugar water in PC group and EEA intervention groups was significantly higher than that in DD group (P < 0.05).

As shown in Fig. 4B (FST results), DD mice showed a longer immobility time (recorded as the absence of escape-oriented behavior in the FST) compared to that in the control mice (P < 0.05). After 4 weeks of treatment, for mice treated with EEA, the immobility time in the experiment was remarkably shorter compared to that in the model group (P < 0.05).

In Fig. 4(C and D) (OFT results), at the same time, compared with NC group, the total movement distance of open field activity in DD group was significantly reduced and the stationary time increased significantly (P < 0.05). Treatment with EEA significantly increased the total distance travelled and reduced the stationary time when compared to the DD group (P < 0.05). These results suggested that EEA could alleviate depression-like behavior in DD mice.

3.3 Effects of EEA on monoamine oxidase and monoamine neurotransmitters in DD mice

The contents of neurotransmitters in the hippocampus after 4 weeks of treatment are shown in Fig. 5(B, C and D). After CUMS exposure, the contents of neurotransmitters including DA, NE and 5-HT in the hippocampus of DD group mice were significantly lower than those of NC group mice (P < 0.05). Compared to the DD group, treatment with fluoxetine combined with metformin and EEA could increase all these neurotransmitter levels in the hippocampus, and the DA, NE and 5-HT levels in middle dose of EEA group all showed the significantly increase (P < 0.05). Similar results were also observed for monoamine oxidase activity in the hippocampus. Treatment with fluoxetine combined with metformin and EEA could reverse the increase of monoamine oxidase activity in the hippocampus of DD mice caused by diabetes and CUMS (Fig. 5A).

3.4 Effects of EEA on the hyperactivity of the hypothalamic-pituitary-adrenal axis in DD mice

Stress levels are reflected in the amount of CORT, ACTH and CRF hormones in the serum. As shows in the Fig. 6 (A, B and C), compared with NC group, CRF, CORT and ACTH secretion significantly increased in DD group (P < 0.05). However, treatment with fluoxetine combined with metformin and EEA could decrease all these hormones levels. Compared to the DD group, the CRF levels in EEA treatment groups and PC group showed the significantly decrease and the CORT, ACTH and CRF levels in middle dose of EEA group all showed the significantly decrease (P < 0.05). Therefore, even though diabetes and CUMS up-regulated secretions of CORT, ACTH and CRF in DD mice, EEA treatment could modulate the secretion of the three hormones.

3.5 Effects of EEA on oxidative stress in the hippocampus of DD mice

We also detected the changes in the oxidative stress levels in the hippocampus of mice to investigate the antidepressant effects of EEA. Diabetes and CUMS could reduce the level of T-AOC and the content of SOD (Fig. 7A and Fig. 7C), and increase the content of MDA (Fig. 7B) in the hippocampus of mice. Fluoxetine combined with metformin and EEA treatment ameliorate this pathophysiology. As shows in the Fig. 7(A and C), the T-AOC and SOD levels in the EEA-M and PC groups were higher than those in the DD group (P < 0.05). In addition, fluoxetine combined with metformin and EEA treatment could reduce the content of MDA in the hippocampus of DD mice.

3.6 Effects of EEA on neuroinflammation in the hippocampus of DD mice

The pathogenesis of depression is strongly related to neuroinflammatory processes in the hippocampus. The levels of IL-1β, IL-6 and TNF-α were detected by ELISA to assess the effects of EEA on neuroinflammation in the hippocampus. The results showed that the contents of TNF-α, IL-6 and IL-1β in hippocampus of DD group were increased, compared with NC group (P < 0.05). However, after 4 weeks of treatment, the contents of TNF-α, IL-6 and IL-1β in hippocampus of each dose of EEA group were decreased compared with that of DD group (P < 0.05) (Fig. 7D, Fig. 7E and Fig. 7F). These results indicated that diabetes and CUMS could aggravate neuroinflammation in the hippocampus, but EEA treatment could reverse this phenomenon.

3.7 Effects of EEA treatment on hippocampal histology in mice

The hippocampus is the central functional area of the brain. Hematoxylin-eosin staining showed histological changes in the hippocampus. In our study, HE staining and Nissl staining were used to observe the histopathological status of CA1 regions in the hippocampus of mice. As shows in the Fig. 8A, normal hippocampal somatic cells were systematically arranged, were regular, had round nuclei and were only lightly stained. However, hippocampal somatic cells of DD group exhibited deep staining of cell bodies, displayed inflammatory cell infiltration. And neuronal cells showed swelling, vacuolar degeneration and necrosis. The hippocampal neurons were atrophic and deformed. In contrast, the hippocampal somatic cells and their nuclei in the PC and EEA treated groups showed lighter staining, and the degree of neuronal damage was also lower. Among them, the hippocampal neurons of the EEA-M group mice were arranged neatly, the gap was normal, and most of the cells had normal morphology.

In the control group, the hippocampus exhibited a normal structure, and the neurons were arranged in a close and orderly fashion; the pyramidal cell layer was thick, and the arrangement was dense and well aligned, with full cell bodies. In the DD group, the density of hippocampal cells was reduced, and the neuronal arrangement was loose, with increased intercellular spaces, loose arrangements, nuclear atrophy, nuclear shrinking in a large number of neurons, and unevenly distributed Nissl bodies. In the PC and EEA groups, the hippocampal structure was normal, the neurons were closely arranged and ordered, the Nissl bodies were evenly distributed, and nuclear shrinking of neurons was less (Fig. 8B).

3.8 Effects of EEA administration on the expression levels of SYN、PSD95、GAP43 in the hippocampus of DD mice

SYN, PSD95 and GAP43 are proteins distributed at different locations of neural synapses, which are closely related to synaptic plasticity. Figure 9 (A, B and C) showed the expression of SYN, PSD95 and GAP43 protein in hippocampal tissue. As shows in the Fig. 9 (D, E and F), compared with NC group, there was significant modulation of SYN, PSD95 and GAP43 expression in DD groups (P < 0.05). Compared with the DD group, the expression level of SYN, PSD95 and GAP43 protein in the CA1 region of hippocampus was increased in the PC group and each dose group of EEA. Taken together, our findings suggested that diabetes and CUMS exacerbates the under-expression of SYN, PSD95 and GAP43 proteins in DD mice. However, EEA treatment could restore abnormal expression of both SYN, PSD95 and GAP43 proteins.

3.9 Effects of EEA on the protein expression of PI3K, p-PI3K, Akt, p-Akt, mTOR and p-mTOR in the hippocampus

In this study, we measured the protein expression and phosphorylation levels of PI3K, Akt and mTOR in the hippocampus of mice (Fig. 10A). The results showed that the protein expressions of p-PI3K, p-Akt and p-mTOR in hippocampus of DD mice were decreased compared with those of NC group (P < 0.05), indicating that the activation of PI3K/Akt/mTOR pathway was inhibited to a certain extent in DD mice. However, as shown in Fig. 10 (B, C and D), levels of p-PI3K/PI3K, p-Akt/Akt and p-mTOR/mTOR were increased in the hippocampus of the EEA treatment group (P < 0.05), compared with the DD group. These results indicated that EEA can significantly increase the phosphorylation levels of PI3K, Akt and mTOR.

Diabetes mellitus is a common metabolic disease, whose pathophysiology is linked to the central nervous system through synaptic transmission, neuroplasticity, inflammatory, and other pathways^{[31, 32]}. Emerging evidences demonstrate that people with diabetes are more likely to have depression and depression exacerbates the pathophysiology of diabetes^{[6, 33]}. Diabetes mellitus associated with depression is a highly harmful disease, but there are still many shortcomings in the current clinical treatment methods^[34]. Therefore, finding effective drugs to treat diabetes combined with depression is one of the future research directions. In this study, we established a mouse model of T2DM combined with depression to evaluate the dual therapeutic effects of AOF, and further explore the mechanism of AOF on diabetes complicated with depression.

Diabetes is characterized by insulin resistance, which is characterized by hyperglycemia and increased serum insulin levels, so blood glucose and insulin levels are important indicators to evaluate diabetes^[1]. In a related research, Yiqiang Xie et al. (2018)^[20] found that AOF treatment decreased blood glucose levels in the T2DM mice, consistent with our findings. HOMA-IR is a key index used to evaluate insulin sensitivity in clinical practice, which can evaluate the body's ability to regulate blood glucose^[35] In our study, the results showed that EEA could reduce fasting serum insulin and HOMA-IR in DD mice. HbA1c is used as the gold standard for observing the long-term glycemic index because it reflects the number of glucose molecules attached to haemoglobin in red blood cells (120 days)^[36]. Glucose tolerance can reflect the ability to regulate the changes in blood glucose^[37].Our results suggested that EEA could reduce HbA1c levels and improve glucose tolerance impairment caused by diabetes and CUMS in DD mice. On the whole, EEA may regulate glucose metabolism and reduce blood glucose in DD mice by regulating insulin level and improving insulin sensitivity.

Hippocampus is an important part of the brain that regulates emotions, memory and cognitive functions. Meanwhile, it is the main target for brain-nerve injury caused by diabetes^[38]. Diabetes and depression are biologically related. Both diseases damage the structure and function of the hippocampus^[10]. Past research has demonstrated that diabetic and CUMS rats exhibit significant depression-like behavior^[39].This is consistent with our findings. Our results suggested that diabetes and CUMS can result the changes in hippocampal morphology and function of DD mice and induces reductions in excitability, activity, cognitive abilities, and sensitivity to rewarding stimuli. The results of this study not only demonstrated the association between depression-like behavior and hippocampal injury, but also demonstrated that EEA could reverse hippocampal injury and improve depression-like behavior in mice.

Oxidative stress is caused by the imbalance between ROS and antioxidants, which can cause oxidative damage to lipids, proteins and DNA and even cause cell apoptosis^{[40, 41]}. The brain is rich in lipids and consumes a lot of oxygen to function properly, so it is vulnerable to oxidative stress. Reduced activity of antioxidative enzymes and increased levels of oxidative stress products in the hippocampus have been reported in patients with depression^[42]. And animal studies have confirmed that eliminating excessive oxidative stress could alleviate depressive symptoms^{[43, 44]}. In this study, SOD and T-AOC levels were decreased and MDA activity was increased in the hippocampus of diabetic mice exposed to CUMS. These changes were reversed by EEA treatment. These results indicated that EEA could regulate the oxidative imbalance of hippocampus and may have the potential to repair hippocampus injury.

Typically, low levels of proinflammatory cytokines are present in the CNS and act as modulators of neural function and neuronal survival. However, in different models of depression, proinflammatory cytokines are highly expressed in the brain, leading to impaired neuronal regeneration and neurotrophic factor release^[45]. Thus, cytokines such as IL-1β, IL-6, and TNF-α have been shown to be the key mediators leading to depressive-like behaviors and can even be used to predict subsequent depressive symptoms^{[46, 47]}. In this study, EEA treatment reversed the increased expression of IL-1β, IL-6, and TNF-α in the hippocampus of mice induced by diabetes and CUMS. This result indicated that EEA treatment could reduce the production of inflammatory factors and thus regulate neuroinflammation.

The hippocampus is the central architect of hypothalamic-pituitary-adrenal axis, which regulates emotion, memory and cognitive behavior. Unfortunately, chronic stress could increase glucocorticoid levels, impair the normal structure of hippocampal neurons and reduce their density^[39]. The HPA axis is continually hyperfunction, which continuously increases CORT levels. High glucocorticoid levels can cause lasting alterations to the plasticity and structural integrity of the hippocampus, and this mechanism may contribute to the development of depression^[48]. In addition, evidences demonstrate that the Glucocorticoid negative feedback is impaired in T2DM patients resulting in HPA axis hyperactivity and hypercortisolism. And the hyperfunction of HPA axis leads to increased glucocorticoid secretion from the adrenal gland, leads to insulin resistance in peripheral target tissues, and aggravates diabetes^{[49, 50]}. This may be one of the reasons for the easy comorbidity of diabetes and depression. Our results indicated that the serum levels of CORT, ACTH and CRF are increased in diabetic mice exposed to CUMS, which may be one of the key factors in the progression of diabetes and depression. However, EEA reversed this change, reducing the serum levels of CORT, ACTH and CRF in DD mice. This also suggested that EEA may treat diabetes and depression by attenuating HPA axis hyperfunction.

The occurrence and development of depression is related to monoamine neurotransmitters. 5-HT, NE and DA are the most important neurotransmitters in the pathogenesis of depression^[51]. MAO is an enzyme protein, mainly present in the human nervous system and liver, which can catalyze the oxidative metabolism of monoamine neurotransmitters. Its elevated activity will cause the decomposition of monoamine neurotransmitters to be enhanced, resulting in a decrease in the content of monoamine transmitters in the synaptic gap, which plays an important role in the occurrence and development of depression^[52]. In this study, the results showed that diabetes and CUMS significantly decreased the contents of DA, NE, 5-HT in the hippocampus of mice, which suggested that dysfunction of the DA, NE and 5-HT systems and obstruction of the metabolic pathway occurred in DD mice. In contrast, EEA treatment reversed the above changes and decreased the activities of MAO. This suggested that EEA could improve the depressive symptoms of mice by decreasing MAO activity and increasing the secretion of neurotransmitters.

Synaptic plasticity refers to the phenomenon of persistent changes in synaptic morphology and function. It is one of the most basic and important functions of the brain and it is the ability to perceive, evaluate and remember complex information, and make adaptive responses to stimuli^[53]. Previous study has shown that decreased synaptic activity in the hippocampus may lead to poor binding of neurotransmitters to synapses, which is one of the causes of depression in mice^[54]. As a postsynaptic component marker, PSD95 is not only the key protein for dendritic spine formation, but also the key for synaptic maturation and synaptic function^[55]. SYN is a specific marker protein located in the presynaptic membrane. It is considered as a marker of synaptic plasticity because of its participation in the synapse formation of neurons and it can be used as one of the indicators to detect the density and distribution of synapses^[56]. GAP-43 is a specific phospholipid protein, which exists on the membrane of nerve cells and is closely related to nerve regeneration, development and synaptic plasticity^[57]. In this study, we found that the expression of SYN, PSD95 and GAP-43 in the hippocampus in DD mice was significantly decreased, and EEA could significantly increase the expression of SYN, PSD95 and GAP-43 proteins in the hippocampus of DD mice. This demonstrated that EEA may play an antidepressant role by regulating the synaptic connections in the hippocampus of mice, increasing the combination of synapses and neurotransmitters such as 5-HT, NA and DA.

The PI3K/Akt pathway is an important mediator in signal transduction^[58]. It is well known that PI3K belongs to a conserved family of signal transduction enzymes that regulates a variety of cellular processes, such as inflammation, apoptosis, and cell activation^[59]. PI3K plays important roles in cell growth, survival, differentiation, glucose transport and metabolism. The important roles of PI3K subunits in regulating glucose and lipid metabolism have also been revealed^[60]. The serine/threonine kinase AKT, a major downstream molecule of the PI3K signaling pathway, is expressed in different tissues, such as brain and testis. AKT is expressed in different isoforms in specific tissues and plays the key role in inflammation or the maintenance of physiological functions of organs^[61]. mTOR is a serine/threonine protein kinase, which belongs to the PI3K-associated kinase protein family that participates in regulating cell proliferation, death, survival, and protein synthesis^[62]. The PI3K/Akt/mTOR pathway play the pivotal role in depression^[63]. Studies have found that the PI3K/Akt/mTOR pathway has certain impacts on synaptic plasticity, synaptic morphology and transmission efficiency, and is involved in the regulation of hippocampal neurogenesis^{[64, 65]}. Many studies have shown that PI3K/Akt/mTOR pathway plays an important role in the antidepressant effects of drugs^{[63, 66]}. These findings suggested that targeting PI3K/Akt pathway may be used for alleviating depression. In the current study, we found that EEA treatment promotes the phosphorylation of PI3K, Akt and mTOR proteins in the hippocampus of DD mice, suggesting that EEA may improve diabetic and depression by activating PI3K/Akt/mTOR pathway to improve synaptic plasticity and promote hippocampal neuron regeneration.

This experiment showed that EEA could improve glucose metabolism and depressive behavior in DD mice. EEA could also ameliorate hippocampal damage, inhibit oxidative stress, reduce neuroinflammation, inhibit HPA axis, regulate neurotransmitter levels and improve synaptic plasticity. The ethanol extract of AOF could also improve diabetic depression by activating PI3K/Akt/mTOR pathway. Therefore, this study provides evidence for the pharmacological effect and mechanism of AOF on improving diabetic depression, and provides new scientific theoretical basis for the future development of AOF and the prevention and treatment of diabetes complicated with depression.

Funding

This work was supported by the National Natural Science Foundation of China (31372453) and the Major Collaborative Innovation Project of Nanyang City, Henan Province, China (21XTCX12005).

CRediT authorship contribution statement

Qilun Zhou: conceptualization, investigation, visualization, data curation, writing – original draft, writing – reviewing and editing. Yue Qi: investigation, visualization, formal analysis, data curation. Jinlan Deng: investigation, visualization, formal analysis, data curation. Ruonan Li: investigation, visualization. Yongping Zhang: data curation. Xiaofeng Zhang: supervision, writing – reviewing & editing.

Acknowledgements

We would like to thank the platform provided by the College of Public Health, Zhengzhou University.

Data Availability Statements

The data sets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethics Approval All animal procedures were performed in accordance with the National Research Council's Guide for the Care and Use of Laboratory Animals.

Consent to Participate Not applicable.

Consent for Publication Not applicable.

Conflict of Interests The authors declare that they have no known competing financial interests or personal relationships.

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Ethanol extract of Alpiniae oxyphyllae fructus regulates glucose metabolism, the HPA axis and hippocampal function in diabetic mice with depression

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Abstract

Figures

1 Introduction

2 Materials and methods

2.1 Chemicals

2.2 Plant materials and preparation of ethanol extract of AOF (EEA)

2.3 Animals

2.4 Experimental design

2.5 Oral glucose tolerance test (OGTT)

2.6 Behavioral Tests

2.6.1 Open Field Test (OFT)

2.6.2 Sucrose preference test (SPT)

2.6.3 Forced swimming test (FST)

2.7 Biochemical measurements

2.8 The histopathology of the hippocampal tissues

2.8.1 Hematoxylin and eosin (H&E) staining

2.8.2 Nissl staining

2.9 Immunohistochemistry (IHC)

2.10 Western blot (WB) analysis

2.11 Statistical analysis

3 Results

3.1 Effects of EEA on body weight and glucose metabolism in DD mice

3.2 Effects of EEA on depressive behavior in DD mice

3.3 Effects of EEA on monoamine oxidase and monoamine neurotransmitters in DD mice

3.4 Effects of EEA on the hyperactivity of the hypothalamic-pituitary-adrenal axis in DD mice

3.5 Effects of EEA on oxidative stress in the hippocampus of DD mice

3.6 Effects of EEA on neuroinflammation in the hippocampus of DD mice

3.7 Effects of EEA treatment on hippocampal histology in mice

4 Discussion

5 Conclusion

Declarations

References

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