Diabetes, an alarming modern-day epidemic, imposes a substantial burden due to its rising incidence rate. Diabetes, in addition to its metabolic consequences, has emerged as a contributor to cognitive decline via a number of underlying processes, including neuroinflammation. Among the critical processes linked to diabetes, the release of pro-inflammatory cytokines has received a lot of attention. The aim of this current research study is to determine the potential of 6-amino flavone as an agent for treating Alloxan-induced diabetes mellitus and neuroinflammation in albino mice. The anti-diabetic and anti-neuroinflammatory effects were assessed through Y-maze, Morris water maze, and probe test in all four groups. The results indicated that the 6-amino flavone group showed a promising in-crease in short-term and long-term cognitive functions compared to the alloxan-induced group. Noticeably, the western blot analysis depicted that 6-amino flavone exerts antioxidant, anti-diabetic, and neuroprotective effects by inhibiting the p-IRS/TNF-α signaling pathway and enhancing the synapse performance via upregulation of SYP and PSD-95 proteins in the Allox-an-induced diabetes mellitus group. The results of the current study infer 6-amino flavone as a potent therapeutic drug against diabetes complications and associated neuroinflammation.
Research Article
Therapeutic Potential of 6-Amino Flavone: A Novel Approach for Diabetes and Inflammation via TNF-α/ p-IRS Signaling Pathway in Albino Mice
https://doi.org/10.21203/rs.3.rs-3991833/v1
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Albino Mice
6-Amino Flavone
Diabetes
Diabetes mellitus, often known as type-2 diabetes, is a lifestyle illness that affects individuals all over the world. It is a disorder where the body's glucose metabolism is hampered, causing a rise in blood glucose levels 1. Type 2 diabetes mellitus (T2DM) can result from the body developing insulin resistance or from the pancreatic beta cells not producing enough insulin. When the beta cell size and function of the pancreas reduce, the cells of the body fail to compensate for a high quantity of insulin secretion because of insulin obstruction, which proves that patients with T2DM can make insulin but their body cells cannot use it. Type 2 diabetes also possesses a wide variety of adverse effects on the structural and functional integrity of the brain 3.
Two of the paramount pathophysiological processes, peripheral insulin resistance, and defective pancreatic islet β-cell insulin secretion, which are the characteristic features of diabetes mellitus, are linked with inflammatory signaling cascades 4. Though the etiology of inflammation is different from that of the pancreatic islet cells, the fundamental mechanisms of insulin resistance are associated with that of an inflammatory response 5. When the progression of islet β-cell starts to fail, it causes the proliferation of β-cells together with pancreatic islets hypertrophy and as a result, an inflammatory response is triggered which initiates fibrosis of islets and apoptosis for the reduction of cells 6. In diabetes, hyperglycemia is referred to as the critical upstream mechanism and henceforth as a consequence, triggering micro-inflammation as a downstream mechanism 6. The contribution of inflammation to insulin resistance can be mapped by combining the innate immune system with metabolism through a nutrient-sensing pathway. Similar to the pathogen-sensing pathways, nutritional elements use collective receptors for signaling pathways thus activating adipocytes and macrophages via common receptors e.g. toll-like receptors 7.
Inflammation is a predominant element associating nutritional excessive overload with insulin resistance and elevated visceral adipocyte mass 8. When the signaling cascade insulin binds to its receptor during an insulin-sensitive stage, tyrosine-residues of the insulin receptor substrate 1 (IRS-1) phosphorylates and result in the downstream of insulin signaling 9. On the other hand, during the insulin-resistance stage, numerous serine kinases like JNK, NFκB kinase inhibitor, and ribosomal protein S6 kinase are activated by the pro-inflammatory cytokines 10. As a result, during the insulin-signaling pathway, the action of insulin is inhibited due to the activation of these kinases which occurs due to the phosphorylation of serine-residues rather than tyrosine-residues 9. The progress of insulin resistance is characterized by JNK and IKKβ/NF-κB transcription factor-signaling pathways 11. The initiation of both pathways involves activators and up-regulators of NF-κB. Toll-Like Receptors (TLRs) and receptors further aid the activation of these pathways for Advanced Glycation End Products.
The human diet contains flavonoids, which are physiologically necessary polyphenols that are extensively dispersed in higher plants. They have a wide spectrum of pharmacological and biological actions, including anti-cancer, antioxidant, anti-allergy, and anti-inflammatory effects. Flavonoids are a key component for the synthesis of numerous therapeutic drugs due to the fact that they are easily isolated, prepared, and distributed therefore, play a vital role in drug design and discovery 13. Flavonoids inhibit the instigation and development of neurodegeneration by inhibiting the neuronal apoptotic cell death instigated by neuro-inflammation, oxidative stress, and other pathological hallmarks of neurodegenerative diseases 14. Although, many different types of flavonoids have been tested and proven effective to slow down the progression of disease with different chemotherapeutic approaches, 6-amino flavone, however, has not previously tested to act as a therapeutic agent in diabetes mellitus. The current research study was, therefore, designed to assess the therapeutic potential of 6-amino flavone by stimulating the TNF-α/p-IRS signaling pathway to inhibit neuro-inflammation and memory dysfunction in mice models for induced diabetes mellitus.
2.1. Chemicals
Alloxan purchased from Santa Cruz (CAS 2244–11 − 3), 6-Aminoflavone purchased from sigma Aldrich (CAS 4613–53 − 0), Tris - based lysis buffer T - PER [Thermo Fisher CAS No.: 78510], Bio-Rad Protein Assay dye [Bio-Rad CAS No.: 5000006], Laemmli sample buffer (2x) [Bio-Rad CAS No.: 161–0737], Tris base [Sigma-Aldrich CAS No.: 77–86 − 1], Ethanol [Sigma-Aldrich CAS No.: 64–17 − 5], Acrylamide [Sigma-Aldrich CAS No.: 64–17 − 5], Bisacrylamide [Sigma - Aldrich CAS No. : 868–63 − 3], Sodium dodecyl sulfate (SDS) [Sigma-Aldrich CAS No.: 151–21 − 3], Ammonium persulphate (APS) [Sigma-Aldrich CAS No.: 7727–54 − 0], Tetramethyl ethylenediamine (TEMED) [Sigma-Aldrich CAS No.: 110–18 − 9], 20x Transfer buffer and 1x Tris buffer saline tween (1x TBST) [Sigma-Aldrich CAS No.: 91414], Enhanced chemiluminescence (ECL) [Bio-Rad CAS No.: 1705061].
2.2. Experimental animal model groups
In the current study, 20 albino adult male mice that were between 30–32 g in weight and around eight weeks old were chosen for the experiment. They were bought from the veterinary research institute in Peshawar. These mice were fractioned into four groups, each group containing 5 mice. These four groups were characterized as follows:
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Control group, with no diabetes and infection.
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Toxic group, to which diabetes was induced by Alloxan.
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Toxic and drug group, to which both Alloxan and 6-aminoflavone were injected.
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Drug group, which was only injected with 6-aminoflavone.
2.3. Drug treatment
The toxic group was treated with Alloxan on alternative days for four weeks. Whereas the toxic and drug group was first treated with Alloxan for four weeks. The third group (Toxic and drug group) was treated with 6-aminoflavone for the next two weeks. The toxic drug and antidote were injected intra-peritoneally.
2.4. Behavioral test
After one month of Alloxan and 6-aminoflavone injections, the behavioral tests were performed as follows to investigate the protective effects of 6-aminoflavone on Alloxan-induced spatial learning and memory deficits in mice.
2.4.1 Y-Maze test
Y-maze is a behavioral test used to observe the short-term memory of mice, this test is performed in a wooden Y-shaped apparatus with three arms at an equal distance of 120⁰. Each arm of the maze was 0.2 m high, 0.5 m long, and 0.1 m wide. In this activity we checked the percentage of the memory of mice of all the groups, if the percentage is higher, it means it has good memory, whereas, the lower the percentage weaker the memory. To train the mice of each group were exposed to the new condition for 10 minutes every two days so that it gets used to it. After the training, the mice of each group were placed one by one within the Y-maze assembly's centre and were allowed to move freely for around eight minutes. The entry of each mouse to the Y-maze’s all the three arms was visually observed and noted. The spontaneous alternation of mice in the arms of Y-maze was noted and calculated by the following formula:
2.4.2 Morris water maze test
The Morris water maze test was performed to examine the hippocampal memory of mice. The apparatus used for MWM test is a huge round tank of about 100 cm in diameter and about 40 cm in height. To perform this test, the tank was filled with water and placed in an adjustable platform. To discover the hidden adjustable platform, first, all the groups of mice were trained by placing them in the tank full of water away from the platform for three consecutive days and then noting the escape latency of the mice for one minute. After the training, the behavioral test was continued for five days and the data was noted. After the performance of the escape latency test the mice were given rest for two days, after the rest, the mice were exposed to a probe test, when the submersed platform was removed in which mice of all the groups were supposed to find the submersed platform and the time they spent in the target quadrant was noted.
2.5. Western blot analysis
Following the behavioral tests, the mice were subjected to sacrifice and the brains of the mice of all the groups were surgically extracted and placed in a separate petri dish to which lateral RNA and PBS solution (1:1) were added for one minute to prevent protein denaturation. After the extraction, through the homogenizer, the brain tissue of each mouse was homogenized separately in 150–200 µl T - PER solution. For protein quantification optical density of each brain sample was measured through a spectrophotometer. For the process of protein quantification, distilled water, and Bio-Rad protein assay dye reagent were used with a 4:1 concentration, and the optical density was measured at 595 nm wavelength.
Afterward, western blot analysis was conducted using the methods that were previously described with some slight optimizations and modifications 15. SDS-PA Gel Electrophoresis (10%) was performed for the assessment and quantification whilst loading the equal quantity (30 µg / sample) of sample protein and a pre-stained protein marker ranging from 10–245 kDa was loaded onto a 10% SDS PAGE stacking gel under optimized environment. The protein marker was used to find out the molecular weight of the mentioned peptide. Afterward, the peptides were shifted to a polyvinylidene difluoride (PVDF) membrane via a turbo transblot (Bio-Rad). We utilized four distinct mouse-derived antibodies (TNF-α, p-IRS, SYP, and PSD95) from Santa Cruz Biotechnology, Santa Cruz, CA, USA, diluted at 1:1000 in 1x TBST, and applied them to a PVDF membrane at 4°C for 24 hours to detect our specific protein of interest. Furthermore, to avoid unnecessary protein interaction without any specific site, the PVDF topology was obstructed with a 5% skimmed milk solution 15. The rinsed blots were then protected at standard temperature with anti-mouse 2nd line IgG-HRPs for two hours. Lastly, the results were developed on x-ray sheets and were visualized by ECL (enhanced chemiluminescence) detection reagent. Then x-ray sheets were examined and computerized Sigma-Gel-program (SPSS, Chicago, IL, USA) was utilized to find the optical densities of the desired proteins. The list of all the antibodies used together with their catalog numbers (Table 1).
| S. No | Antibodies | Catalog No. |
|---|---|---|
| 1 | TNF-α | sc-52746 |
| 2 | p-IRS | |
| 3 | SYP | |
| 4 | PSD-95 | |
| 5 | IgG-HRPs | sc-2031 |
2.6. Bio-statistical analysis
All the statistical analyses were done as reported earlier 15 with slight modifications. Scan statistics were analyzed utilizing specialist computer-based tools, i.e. Image-Java for bands density examination, GraphPad Prism 8, Illustrator-Photoshop, etc. The density of peptides was expressed in arbitrary units (A.U) and the statistical ± S.E.M. # shows a major difference from the values of the control group, while * shows a major differences from the values of the albino-mice; *#P 0.05, **## P 0.01, and ***### P 0.001.
2.7. Ethical Declaration
Our research followed rules for taking care of animals and got approval from the Animal Ethics Committee at Sarhad University in Pakistan. We used albino-mice because they can help both people and animals, and we tried our best to use as few mice as possible and make sure they didn't suffer too much.
3.1 Alloxan mediated oxidative stress in the brain of the mice
Alloxan causes oxidative stress when administered to adult mice which then signals P-IRS to increase its phosphorylation due to which TNF-α also increases (TNF-α is a small protein used by the immune system for self-signaling due to which inflammation is caused in pre synaptic and post synaptic neurons). Due to this reason, pre synapse that is transmitting messages, and post synapse that is receiving messages does not take place, as a result, the synapse is affected, hence, the hippocampus of the mouse is affected which results in memory impairment.
Figure (1a) shows the actual picture of the X-ray in which the band is narrow in the control group, which means secretion of P-IRS in control is less. In Alloxan induced group the band gets broader, in the 3rd group of Alloxan plus 6-aminoflavone, the band gets narrower again which indicates that 6-aminoflavone has decreased the secretion of P-IRS. When 6-aminoflavone was injected into a completely normal mouse to which no Alloxan was injected shows a completely less secretion of P-IRS as compared to all groups and the same happens in the case of TNF-α. The bands of β-Actin show the amount of protein that was taken for each group in an equal amount (Fig. 1).
3.2 6-AminoFlavone Stimulated Synaptic Assessment Against Alloxan Induced Oxidative Stress in the Mice’s Brain
6-aminoflavone reverses the effect of Alloxan as it decreases the P-IRS phosphorylation due to which TNF-α is no more signaled, hence pre synaptic and post synaptic neurons accept no more inflammatory response due to which memory impairment caused by Alloxan is improved by 6-aminoflavone. Figure 2 shows the synaptic activity of all the groups. In the control group, the synaptic band is normal when Alloxan is injected the synaptic activity has decreased and the band gets narrower, after injecting 6-aminoflavone the band gets broader which shows improvement in the synaptic activity. In the last group, the normal mice to which only 6-Aminoflavone is injected show the broader band which proves 6-aminoflavone can improve the synaptic activity. The same happens in the case of PSD-95 with the same amount of β-Actin for all the groups.
3.3 6-Aminoflavone enhances memory and behavior in Alloxan induced diabetic mice
3.3.1 Y-Maze Examination
The behavior of experimental mice in a novel environment was observed by Y-maze examination. For 4 experimental groups the percentage of spontaneous alternation was calculated through the formula (% of spontaneous alteration = No of triplicates / T. No of arm entries × 100). Y-maze test proved that the normal mouse had a high percentage of spontaneous alternation while the Alloxan induced mouse had a low percentage of spontaneous alternation, but the 3rd group injected with both Alloxan and 6-aminoflavone had a higher percentage of spontaneous alternation as compared to the group of mice injected only with Alloxan. The difference in the percentage of spontaneous alternation of mice in the Y-maze observation is shown in Fig. 3.
3.3.2 Morris Water Maze Observation
Morris water maze observation evaluates the outcome of 6-aminoflavone on spatial learning in Alloxan induced diabetic mice. For this test, the mice were first trained for three days consecutively and then were given rest. After the rest the different four groups of mice were exposed to the Morris water test for 5 days and the data was recorded. The results concluded that the mean latency was decreased in all four groups to identify the adjustable platform during the training period.
On day one, the Alloxan induced mice found the platform within 60 seconds; while the group induced with both Alloxan and 6-aminoflavone found the platform within 30 seconds. On 2nd day Alloxan induced mice found the adjustable platform in less time that is 50 seconds, whereas, the Alloxan plus 6-aminoflavone group found the adjustable platform within 26 seconds, likewise the time of finding the adjustable platform decreased on the 3rd, 4th, and 5th day. This test proved that 6-aminoflavone has the potential to treat the spatial learning that was impaired by Alloxan. The escape latency of all the groups was lowered day by day and was almost the same as the control group as shown in Fig. 4.
3.3.3 Probe Test
On day 6 the probe test was conducted by removing the adjustable platform. The time taken to find the stand by all 4 groups was observed in the target quadrant. This test proved that Alloxan induced mice spent less time in the target quadrant as compared to the control group that spent more time. The mice group induced with both Alloxan and 6-aminoflavone spent more time in the target quadrant as compared to the Alloxan induced mice, but the time spent by this group was less than the control mice. Figure 5 shows data for the probe test performed during the MWM test in which the adjustable platform was removed.
3.3.4 Random Glucose Test
The glucose level of the mice was checked through a random glucose test. The mouse of the control group showed normal blood glucose levels i.e. between 60 to 80 mg / dl, after injecting Alloxan diabetes was induced and blood glucose level was found around 230 mg / dl, after injecting 6-aminoflavone in the diabetic mouse, a great decrease was noticed and the blood glucose level dropped to 120 mg / dl which proves 6-aminoflavone to be one of the promising therapeutic agents for diabetes (Fig. 6).
By altering glucose transfer to the brain and lowering glucose metabolism, diabetes can in some situations; such as metabolic irregularities, increase the incidence of Alzheimer's disease. Furthermore, the metabolism of the brain places severe demands on mitochondria because of its high-energy consumption and abundant lipid content. Thus, compared to the rest of the body, the brain may be more vulnerable to oxidative damage. New research reveals that the pathology caused by amyloid-(A) is influenced by both oxidative stress and mitochondrial dysfunction 16. The majority of dietary plants, including fruits, vegetables, and cereals that are commonly eaten in the human diet, contain bioactive chemicals called flavonoids, which have one or more aromatic rings containing hydroxyl moieties. Plants manufacture these secondary metabolites to defend themselves against ultraviolet radiation, oxidants, and diseases caused by bacteria, fungi, and viruses 17. Several flavonoids from the flavone family have been discovered to have positive effects on diabetic retina. For instance, after laser photocoagulation, apigenin administration showed a substantial decrease in choroidal neovascularization. Additionally, it reduced endothelial cell migration and proliferation and controlled endothelial cell function 18. Flavonoids have been found to prevent the onset and progression of neurodegeneration by preventing neuronal apoptotic cell death triggered by neuroinflammation, oxidative stress, and other pathogenic features of neurodegenerative illnesses 19. Flavones are utilized in the treatment of cancer, cardiovascular disease, and neurological illnesses. They also possess anti-proliferative, antioxidant, anti-tumor, acetylcholinesterase, estrogenic, and anti-inflammatory properties. Since oxidative stress is thought to be the root cause of the majority of metabolic diseases, recent research must demonstrate the protective effects of flavones against oxidative stress-related illnesses 20. One of the most common structural changes made to flavonoids was the addition of amino groups to the flavone scaffold 21. Amino group-containing derivatives of flavones were investigated as potential anticancer agents 22. The first of these to begin human clinical trials was flavopiridol, a cyclin-dependent kinase inhibitor 23.
Pathological changes in the central nervous system (CNS) are linked with diabetes mellitus (DM), which results in cognitive and motor deficiencies, as well as an increased risk of brain vascular problems 16. Diabetes mellitus was induced in this study by injecting 60 mg/kg of body weight alloxan intraperitoneally. Alloxan causes diabetes by killing pancreatic beta cells, which are responsible for the production, storage, and secretion of insulin, a peptide hormone that regulates glucose and causes hyperglycemia in mice 24–26. It is also specifically harmful to pancreatic -cells of the Langerhans islets by inducing necrosis. It has two different pathogenic effects: selective reduction of glucose-induced insulin secretion via glucokinase inhibition, and the production of free radicals 27, 28. Insulin receptor substrates-1 (IRS-1) and inflammatory factors such as TNF- and free fatty acids (FFAs) are downstream signaling molecules that have been identified as potent mediators of insulin resistance associated with obesity. These molecules exert their effects on IRS − 1 serine phosphorylation 29, 30. Figure 1 shows that P - IRS expression is highest in Alloxan induced group while its expression is least in the control group. The group of mice that received alloxan along with 6-aminoflavone showed less expression of P - IRS. This indicates that 6-aminoflavone has lowered P - IRS expression. A similar pattern of expression was seen for TNF - α in these respective groups. Because Alloxan raises glucose levels in other sensitive organs like the brain, it can be used to cause glucose-related memory deficits 31. Following high-frequency stimulation of afferent neurons, long-term potentiation (LTP) occurs, which increases synaptic strength. It has been studied extensively as a sort of synaptic plasticity that suggests a cellular learning and memory mechanism 32. In this study, improvement in the expression of presynaptic protein Synaptophysin (SYP) and postsynaptic density protein 95 (PSD 95) in the 6-Amino Flavone treated group was observed, which shows its importance as a neuroprotective agent against memory impairment.
Metabolites such as glycosides, alkaloids, and flavonoids have been discovered in the majority of plants with hypoglycemic characteristics 33, 34. This study also discovered that 30 mg/kg body weight of 6 - Amino Flavone lowered blood glucose levels in mice.
Insulin in the brain is involved in energy balance, learning, and memory, besides its other functions. In the meantime, insulin can influence neurotransmitter release, neuronal survival, and synaptic plasticity directly 35. Studies have reported that disturbances in brain insulin signaling pathways have been linked to biochemical, molecular, and histopathological changes in Alzheimer's disease 36. In the novel object recognition task and the water maze test, however, 6-Amino Flavone treatment improved behavioral deficits. These types of behavioral tests are frequently used to evaluate memory and learning abilities 37–39. The present study infers that 6-Amino Flavone appears to have a neuroprotective effect on cognitive performance.
The results indicated that the 6-amino flavone group showed a promising increase in the short term and long-term cognitive functions compared to the alloxan induced group. Noticeably, the western blot analysis depicted that 6-amino flavone exerts antioxidant, anti-diabetic, and neuroprotective effects by inhibiting the p-IRS/TNF-α signaling pathway and enhancing the synapse performance via upregulation of SYP and PSD-95 proteins in the Alloxan-induced diabetes mellitus group. The findings of the present study infer that 6-amino flavone could be used as a potential therapeutic agent for treating diabetes complications and associated neuroinflammation.
Ethical approval
The present investigation was conducted according to ethical principles of animal careand was approved by the Animal Ethical Committee, Department of Biochemistry, Abdul Wali Khan University, Mardan, Pakistan for studies involving animals.
Informed Consent Statement
Not applicable.
Data Availability Statement
No data was used for the research described in the article.
Conflicts of Interest
The authors declare no conflict of interest.
Funding
The study was funded by the Deputyship for Research and Innovation, Ministry of Education in Saudi Arabia (Project number: ISP23-81).
Acknowledgement
The authors extent their appreciation to the Deputyship of Research and Innovation, Ministry of Education in Saudi Arabia for funding this research work through the project with number: ISP23-81.
Author Contributions
Conceptualization, VS, AK and AW Methodology, ZA, HAA, RMA, MH, PT and SAS. Formal analysis, WF, RMA, AK, SAS and AW Investigation, VS, HAA, WF, AK and PT Writing—original draft preparation, ZA, MH and AW writing—review and editing, VS, HAA, RMA, AK, PT, SAS and AW supervision, VS, AW and AK. Project administration, AW and VS. Funding acquisition, VS and AK. All authors have read and agreed to the published version of the manuscript.
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No competing interests reported.