Rosmarinic acid inhibits DNA glycation and modulates the expression of Akt1 and Akt3 partially in the hippocampus of diabetic rats

Non-enzymatic glycation of DNA and the associated effects are among pathogenic factors in diabetes mellitus. Natural polyphenols have anti-diabetic activity. Herein, the protective role of one of the phytochemicals, rosmarinic acid (RA), was evaluated in glycation (with fructose) of human DNA and expression of Akt genes in the hippocampus of diabetic rats. In-vitro studies using fluorescence, agarose gel electrophoresis, fluorescence microscopy, and thermal denaturation analyses revealed that glycation causes DNA damage and that RA inhibits it. In-vivo studies were performed by induction of diabetes in rats using streptozotocin. The diabetic rats were given RA daily through gavage feeding. The expression of Akt genes (inhibitors of apoptosis) in the hippocampus was evaluated using RT-qPCR. In diabetic rats, Akt1 and Akt3 were significantly down-regulated compared to the control group. Treating the diabetic rats with RA returned the expression of Akt1 and Akt3 relatively to the normal condition. Past studies have shown that diabetes induces apoptosis in the hippocampal neurons. Given that glycation changes the genes expression and causes cell death, apoptosis of the hippocampal neurons can be due to the glycation of DNA. The results also suggest that RA has reliable potency against the gross modification of DNA under hyperglycemic conditions.


Results
Glycation and UV-vis spectroscopic characterization of DNA. The absorption spectroscopic analysis of the fructose-modified DNA showed a broad absorption band centered at 260 nm with marked hyperchromicity after 15 days of incubation; this was comparable to the native form (Fig. 1). Upon glycation, the trend of kinetic changes at 260 nm kept on rising within 5, 10, and 15 days of incubation in the PBS (pH 7.4). Compared to the 260 nm peak of the native DNA, the hyperchromicities shown by 15 days was 29.11%. Furthermore, the ratio of the intensities at 260:280 nm (A260/A280) was decreased for DNA in the presence of fructose (Table 1).
Agarose gel electrophoresis. Agarose gel electrophoresis was used to determine the fate of DNA during fructation. Figure 2 demonstrates the electrophoretic analysis of the DNA incubated with fructose when RA was present. The difference in the band pattern and intensity was exceptionally informative. Electrophoretic migration of the native DNA reflected the pattern of the typical genomic DNA which was comparable to the migration pattern of the ligand-treated and modified DNA (Fig. 2). DNA sample not exposed to fructose migrated as a sharp single band initially with the appearance of an additional minor band, whereas the disappearance of the initial band was evident for the fructose-modified DNA (lane 1-3) even after 5 days of exposure. No remarkable changes were observed at the band intensities of the treated samples with RA (lane 7-9); meanwhile, for the samples exposed to fructose at a longer duration (10-and 15-days), their protective effect was substantial  Fluorescence studies. Glycation causes AGEs to form in DNA along with additional fluorescent adducts that establish disrupt human genes 21 . DNA-AGEs were measured by fluorescence intensity at the excitation/ emission wavelength of 370/440 nm. The results obtained by fluorogenic AGEs experiments showed a great extent of alteration in the DNA structure after adding fructose ( Fig. 3 and Table 1). It is worth noting that an interesting phenomenon occurred; about 43% of fluorescence intensity was diminished in the treatment with RA (25 µM) (Fig. 3).

Fluorescence microscopy.
To further explore the binding nature of fructose with DNA and evaluate the RA influence, a visual inspection experiment was applied via fluorescence microscopy. As could be seen from the images of Fig. 4, after 15 days of incubation, amorphous-like aggregates were detected in the samples containing DNA-fructose (Fig. 4B), which was comparable to the control (Fig. 4A). Interestingly, after 15 days of incubation, these insoluble aggregates were markedly diminished by the presence of RA (Fig. 4C).
Zeta potential measurements. The zeta-potentials of DNA, Fructose-DNA, and the sample treated with RA were − 3.5 mV, − 0.5 mV, and − 1.8 mV, respectively ( Fig. 5 and Table 1). The results of the particle size distribution of RA complexes were still in the nano-size region.
Thermal denaturation. Thermal denaturation analysis was performed to address the impact of glycation.
Based on thermal denaturation analysis, the melting temperature (Tm) for the native, glycated, and RA treated DNA was 82 °C, 75 °C, and 79.3 °C, respectively (Table 1). Therefore, a decrease was observed in the Tm of the modified DNA.  Downregulation of Akt1 and Akt3 in the diabetic rat hippocampus. Akt1 and Akt3 expression in diabetic rats showed a significant downregulation of 47% (P = 0.0404) and 67% (P = 0.0407), respectively, when compared with the control group (Fig. 7A, C). Treating the diabetic rats with RA returned the expression of Akt1 and Akt3 relatively to the normal condition with P = 0.0231 and P = 0.0236, respectively (Fig. 7A, C). The expression of the Akt2 was not significantly different among the three groups (Fig. 7B).

Discussion
Fructose as an important glycating agent. In hyperglycemia and diabetic conditions, glucose concentration increases in the insulin-independent tissues such as neural tissue, glomeruli, lens, and erythrocytes. Accordingly, the polyol pathway can be activated and sorbitol is converted to fructose by sorbitol dehydrogenase [6][7][8] . The high reactivity of fructose, either directly or through its metabolites, may contribute to the formation of intracellular glycation products and vascular complications 22 . Interestingly, fructose's contribution to the onset of these deleterious reactions is 5-8 times higher than that of glucose 23,24 . Briefly, first, fructose has more reactivity than other sugars. Second, in tissues where the sorbitol pathway is active, glucose is converted to fructose. Third, in diabetic conditions, the amount of fructose in the eye lens may increase by 23 times 25 , making glycation a more likely event in the body. Therefore, in the present study, fructose was used as a glycating agent for these reasons.

DNA glycation and inhibition by rosmarinic acid.
To determine the structural alteration of DNA during the reaction with fructose, UV spectroscopy and fluorescence study were employed. Ultraviolet spectroscopy is often used to determine the conformational changes of chromophores occurring in DNA [26][27][28] . 5, 10, and 15 days after glycation, the trend of the kinetic changes at 260 nm continued to increase. It could be concluded that fructose's modification of nitrogenous bases caused single-strand breakage, which resulted in the destruction of chromophore classes. As a result, the hypochromic phenomenon may be caused by dispersion force interactions between stacked chromophores, which could be explained in terms of extensive strand scission and intensity exchange between different transitions. Moreover, some slight red shifts at the peaks may result from the denaturation-dependent head-to-tail arrangement of the transition dipoles ( Fig. 1). According to Table 1, which represents the difference in the ratio intensities of A260/A280 for the native and glycated DNA, it could be seen how fructation caused marked changes in the structure of the DNA generating light-absorbing molecules.
In the agarose gel electrophoresis study, alteration in the migration profile of fructose-DNA might originate from the significant damage in the amino groups of nucleotides at the advanced glycation stages (15 days incubation), causing further rearrangement and destabilizing the phosphodiester backbone. Modification of DNA by fructose gives rise to breaking single strands and generating nucleotide adducts, due to increased oxidative stress 21,29 . Moreover, the formation of superoxide radicals is involved in the Maillard reaction of DNA with fructose and other sugars 30 . The significant decrease in the single strand breakage for the RA treated samples could be attributed to its established antioxidant activity 19 . Reactive oxygen species (ROS) are a potent mediator causing cellular stress originating from sugars auto-oxidation 31,32 .
Generation of fluorogenic AGEs in the glycated-DNA samples was probed at the excitation/emission wavelength of 370/440 nm. Glycation of DNA by fructose generated fluorescent DNA_AGEs which could be characterized by the emission maxima of 440 nm. This is a typical event of DNA-AGEs that has been detected in vitro in the process of glycation 33 . This finding is also consistent with another study, suggesting that AGEs could be formed as the end-product of DNA molecules, resulting in the single-strand breakage 30 . The quenching effect of RA, as seen in Fig. 3 and Table 1, offers a clue for the reduction of advanced glycation end products during glycation. It suggested that the fructation of DNA resulted in the formation of nucleotide AGEs which could be associated with the increase in the mutation frequency and cytotoxicity 34 .
The results of the fluorescence microscope also confirmed those of the previous experiments. In the samples containing DNA-fructose, amorphous-like aggregates were observed (Fig. 4B); The presence of RA markedly Relative expression of Akt genes in the hippocampus between control, diabetic and diabetic groups treated with RA, by real-time PCR. In the diabetic and treated groups, respectively, there was a significant downregulation of 47% and 32% for Akt1 compared to the control group. (*P < 0.05) (A). Comparison of the relative expression of Akt3, between the control group with the diabetic and treated groups showed a significant downregulation of 67% and 20%, respectively (*P < 0.05) (B). There was, however, no significant difference in the Akt2 gene expression between the three groups (P > 0.05) (C). The columns represent the mean ± SEM. www.nature.com/scientificreports/ diminished these insoluble aggregates (Fig. 4C) However, the reduction of the whole glycation-linked aggregation process by RA might originate from its preserving effect on the global fold of the DNA molecule. The occurrence of reactive Maillard intermediates with DNA may account for the strand scission and increased inter-strand crosslinking, culminating into adducts capable of forming yellow-brown fluorescent compounds 35,36 .
According to the zeta potential results, the particle sizes of the Fructose-DNA, RA-complex were comparable to that of the native DNA (Fig. 5). It is obvious that in the DNA-fructose system in which RA was present, the change in particle size caused by the addition of fructose was much smaller than that of the modified DNA ( Fig. 5 and Table 1). Through these experiments, we found that the interaction of RA made the zeta-potential smaller than that in the fructose-DNA complex and the corresponding particle size obeyed the same trend.
From Table 1, the decrease in the Tm of the modified DNA could be ascribed to the generation of single-strand breaks and/or the altered hydrogen bonding between base pairs. The results were in the same trends of fluorescence intensity values (Table 1) obtained by AGE-specific fluorescence (λex 370 nm, λem 440 nm), thus largely representing the structure of the DNA change after adding fructose and protecting markedly by the applied RA.
Overall, our findings revealed that the fructation of DNA led to the formation of nucleotide adducts with increased oxidative stress. Thus, RA holds a considerable intervention potential with the underlying mechanism of fructose-mediated DNA-AGE formation. The inhibitory action of RA is partially due to its antioxidant potential and ROS scavenging activity; it could also be attributed to stacking with glycogenic nucleotides which direct the ligands to the glycogenic core, thereby counteracting the effect of the formed inter-strand cross-link in the duplex DNA. Since the mechanisms underlying the formation of advanced glycated-DNA may elevate the risk of cancer by enhancing the risk of mutagenesis, our results might help to design nutraceutical-based small molecules useful for decreasing the risk of mutation frequency and other DNA lesions leading to cytotoxicity.

Diabetes, apoptosis in hippocampal neurons.
The previous studies provide evidence that both types of diabetes are associated with functional and structural disorders in the brain [37][38][39] . One of the most critical disorders is apoptosis in hippocampal neurons 9-12 . Significant down-regulation expression of Akt1 and Akt3 (apoptosis inhibitor genes) in the current study also suggested the occurrence of apoptosis in the hippocampal neurons of STZ-induced diabetic rats.
Akt genes with a distinct hippocampal expression. In the hippocampus, each Akt isoform has a distinct expression pattern: Akt1 and Akt3 are predominantly expressed in neurons, while Akt2 is primarily expressed in astrocytes and glia 40 . Akt2 deficiency is associated with insulin resistance, causing a diabetic syndrome with elevated plasma glucose levels; this suggests that Akt2 is involved in the insulin signal 41 . These results, therefore, suggest that the reason for no significant difference in the mRNA level of Akt2 in the present study could be the close relationship between the expression of this gene and insulin and blood sugar levels, as well as insulin signal. It is thought-provoking that astrocytes, unlike neurons, are insulin-dependent, and the present study emphasizes the possibility of DNA glycation in non-insulin-dependent cells.

A link between apoptosis of hippocampal neurons in diabetic conditions and DNA fructation.
Diabetes is associated with oxidative stress due to the increased free radical formation and the decreased activity of the antioxidant defense systems 42 . Hyperglycemia causes increased formation of Reactive Oxygen Species (ROS) through several pathways, including the polyol pathway and glycation 30,43 . Glycation and AGEs change the expression of genes 44,45 , as well as cause cell death 46 . According to these results, as well as those in previous sections, apoptosis in hippocampal neurons may be related to DNA damage induced by the polyol pathway's increased fructose concentration; In other words, apoptosis of the hippocampal neurons can be due to the glycation of DNA. Significant relative return of Akt1 and Akt3 expression to the normal state, based on the treatment of STZ-induced diabetic rats with RA as a natural phenol with the ability to inhibit glycation, confirms the possibility of apoptosis due to the DNA glycation.

Conclusion
The findings of this research, confirm the anti-glycation properties of rosmarinic acid and point to it as a potential biophenol that can effectively minimize diabetes complications. According to the current study, hippocampal nerve cell apoptosis in diabetics may be due to DNA glycation. Therefore, further study into the likelihood of DNA glycation inside diabetic hippocampal neurons and other insulin-independent tissues is suggested to validate this phenomenon.

Materials and methods
All of the chemicals used in this study, whose manufacturer is not listed in the text, are from Sigma Chemical Company, USA. . Three days later, fasting blood glucose levels were determined using tail blood. Only rats were considered diabetic if basal blood glucose levels exceeded 250 mg/dl. After confirmation of diabetes, eight rats in the diabetic group received 30 mg/kg RA (mixed in deionized water), once daily by oral gavage for 8 weeks. This concentration of RA shows anti-oxidant and anti-glycate effects in diabetic rats by reducing the formation of MDA and advanced glycation end products 54 . Rats in the control groups were given an equal volume of water. The animals were anesthetized with a mixture of pentobarbital sodium and phenytoin sodium (Euthanasia III) at the end of week 8. Rats were sacrificed when they failed to respond to a toe pinch. The hippocampus tissue samples were isolated and flash frozen in liquid nitrogen; they were preserved at − 80 °C until further experiments.

Isolation of human
Total RNA extraction and reverse transcription. Total  Statistical analysis. All data were presented as means ± standard deviation (SD). Statistical analysis was performed by one-way ANOVA, using GraphPad Prism software, version 7. A Duncan's post-hoc comparison was then made to analyze the sources of significant differences by SAS, version 9.2. A P-value ˂ 0.05 was considered statistically significant.
Ethical approval. The study was approved by the Ethics Committee of Research, University of Isfahan, Iran (IR.UI.REC.1399.056). Collecting blood samples was performed in accordance with the seventh edition of the Helsinki declaration and its later amendments or comparable ethical standards and the research protocol and written consent was first submitted to and confirmed by the Ethics Committee of Research, University of Isfahan. Written informed consent was collected from each blood donor and the full explanation of the study, including the aims, methods, and sources of funding, institutional affiliations of the researchers, the anticipated benefits, and post-study provisions, was provided to each blood donor. It should be noted that the donors had no diabetes and were over 18 years of age. Also, the present study was approved by the Institutional Animal Care and Use Ethics Committee of the University of Isfahan, Iran, (Ethical number: IR.UI.REC.1399.056) and carried out in compliance with the ARRIVE guidelines. The method of caring for mice as well as their anesthesia is described in the materials and methods section, experimental animals and, samples collection. Table 2. Primer used for the real-time PCR.