Role of rosiglitazone in experimental Wistar rats model of neurodegeneration: Effect of peroxisome proliferator-activated receptors-γ as advanced glycation endproducts receptor of advanced glycation endproducts axis inhibitor

INTRODUCTION

Longevity is the basic stipulation of every living organism. Hence, the food we take and the materials that are being categorised in the eatables need to be thoroughly under watch, as the eatables which are our daily routine intake can be dangerous and can also have the capability to produce reactions within the body and give rise to a hazardous end product as advanced glycation end product. It is not always possible for an ordinary person to keep track of their food intake, as the common man has no track of end products developed inside the body. In the race for survival of the fittest, humans require good health and the proper functioning of all bodily organs to ensure a long and joyful life. Considering neural health, the ministration towards it becomes a foremost priority, and it is also because amyloid beta protein and tau protein, massed in neural cells, can produce neural damage. It is all a popular theory and a scientifically proven fact that the amyloid beta protein is the main culprit for all the ill health related to neurons and neural cells. It is also known that the amyloid beta proteins are generally formed by beta and gamma secretases, which is due to the oxidation process by the reactive oxygen species (ROS) or the free radicals present in our body.^[1] The products that were earlier considered only as the blood sugar regulators (glycation) have an impact on the body and have these changes to produce neural changes. Hence, with these products showing rapid action, the possible treatment would be those drugs that can reduce the increased blood sugar content.

The advanced glycation end products (AGEs) were addressed for the first time by the scientist Louis Camille Maillard. He found a brown-coloured structure being produced in the reaction between carbohydrates and amino acids. The brown-coloured structure formed was the end product.^[2] AGE molecules are formed from a reaction known as the Amadori rearrangement of the fructosamine structures, the product of chemical reactions of aldehyde and ketone with amino groups of the protein structures. These reactions are non-enzymatic and do not require any enzymatic catalyst.^[3] Three stages of end products can be formed – the first and foremost is the reaction between the reducing sugar to produce a Schiff ’s base. The intermediate or mid-stage would be the generation of alpha-dicarbonyl or ox-aldehydes to produce the glyoxal, methylglyoxal and 3-deoxyglucosone.^[4,5] It is firmer and has a stable Amadori structure. Later, rearrangements in the base can develop the most stable structures called advanced glycation end products. The researchers have also found the role of AGE in diseases such as Alzheimer’s, Parkinson’s and amyotrophic lateral sclerosis.^[6,7] While animal studies and research have established a connection between AGEs and neurological conditions, the precise mechanism causing this effect is still unclear. This contributes to the further study of animal histopathology, which would lead to the structures which are additionally being developed. Synapse studies are also required by scientists to understand the effect of AGE in the synaptic gaps and other structures, such as microglia and astrocytes. In this study, some of the parameters are explained with the help of interleukin (IL)-6 and thrombosis necrosis factor-α (TNF-α), furnishing the inflammation structures. On the other hand, in planning for the treatment of such conditions, we have an enzyme, sirtuin-1 (Sirt-1), which is an NAD-dependent deacetylase, which has a function in the ageing and antioxidant processes.^[8,9] The antidiabetic drugs show action on various body organs where the AGE accumulation was the cause of disorders (such as cardiovascular diseases). Discussing the probable mechanism of action of these drugs in neural health, studies have proven their role in neuroprotection and healthy neural cell growth. It is because of its action in decreasing the oxidative stress within the neural cells and preventing neural damage and cell death. Sirt-1 activation and binding enhance the function of externally ingested antioxidants, thus reducing amyloid-β (Aβ) accumulation, and suppress its production. This helps in the treatment of Alzheimer’s disease (AD). In a study of the AD mouse model, Sirt-1 indicates the suppression in the production of Aβ and α-secretase. It is of interest as it can decrease the effects of AGE, as per the recent studies. This could be further tested in laboratories for an extensive study.^[10,11]

The mechanism of Alzheimer’s is still under investigation as many of the researchers and scientists have premises over it, but the conclusion is yet to be drawn. One of the mechanisms is AGE’s involvement in AD.^[12,13] The present animal study aims to show the correlation between them. We try to indicate the influence of AGEs on ROS. The development of AGEs promotes protease-resistant crosslinking that occurs between peptides and proteins. Aβ, tau, α-synuclein and prions are only a few of the proteins associated with neurodegenerative illnesses that are glycated, and the degree of glycation is connected to the patient’s pathological condition.^[14,15] The presence of AGE to such an extent is indicative of its role in neurodegenerative disorders. The activation of nuclear factor kappa (NF-κ) and the presence and accumulation show their involvement in the AGE action. The biochemical assays are indicative that the release of inflammatory mediator’s cytokines, due to increased production of macrophage colony-stimulating factor in neuronal cells, has an impact on the extent of AD, and it is due to the activation of the receptor of AGE (RAGE).^[16,17] The treatment procedure follows the blockage of peroxisome proliferator-activated receptors-γ (PPAR-γ), which uses rosiglitazone in the present study. It reduces the burden produced by the Aβ plaques, reduces the inflammation of the neuronal cells and reduces the conditions of neurodegeneration.^[18,19] PPARs, especially PPAR-γ, can influence the formation and accumulation of AGEs by regulating metabolic pathways. They help improve insulin sensitivity and glucose metabolism, thereby reducing the substrates available for glycation reactions. PPAR-γ activation influences the metabolism of amyloid precursor protein, reducing the production of Aβ peptides, which aggregate to form plaques in AD.

The impact of AGE-RAGE on neurodegeneration has been proven by various articles, and it explains the mechanism of neurodegeneration to be based on the high mobility group box-1, which is a s100 protein family member.^[20] Proteins’ gained structural strength can be altered by almost any kind of post-translational changes that should have at least two significant influences. First, these structural alterations destroy the active pocket of an enzyme, thereby making it less likely for a substrate to bind to its enzyme. Second, hydrophobic clusters within the protein core that are ordinarily hidden are exposed as a result of these changes. Such changes are therefore highly likely to increase the aggregation propensity of proteins, which also leads to the formation of deleterious oligomers. An open carbonyl group is produced at the reaction when the aforementioned carbonyl compounds, AGEs, react with Lys530. This results in oligomers that crosslink by reaction with the oligomer-forming lysines in other polypeptides. These changes ensure that the proteins involved with them have an untoward function.^[21] All these mechanisms are still predictions as the complete clear explanation is unclear. This initiates research on missing links to connect all the possible reasons for neurodegeneration due to AGE-RAGE. In the present study, haloperidol was used as the neurodegeneration-inducing agent, and rosiglitazone was used as the drug to study the effects of PPAR-γ inhibitors in neurodegeneration.

MATERIALS AND METHODS

RESULTS

Physical estimated parameters

The best way to identify a diseased condition is by estimating the body weight, food intake and urine output. In the neurodegeneration induced by haloperidol, swallowing is difficult, and the induced animals reduce their food intake, which also reduces their weight of the patient. The urine output is also reduced as the induced animal faces difficulty in passing urine. These conditions were improved by the rosiglitazone. This can be better understood using the graphical representation of the amount of food intake, body weight of the rat and urine output [Figure 1].

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Figure 1:

Physical estimated parameters, all values are expressed as mean ± SD, when the number of rats used in each group (n = 6). Data were subjected to one-way ANOVA followed by Dunnett’s test when normal saline was compared to the haloperidol control group (disease control), haloperidol and rosiglitazone treated group (Test control) and per se were compared. *a indicates P < 0.0001 when compared to normal saline. NS: Normal saline, DC: Disease control, TC: Treatment control, P: per se.

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Behavioural parameters

The externally observed characteristics of the rats after haloperidol treatment show an increase in weight, an increase in the movement of the rat within the cage and an increase in anxiety. These characteristics were indicative of the neural changes in the rats. The comparison study performed between the rosiglitazone-treated and haloperidol-treated rats shows the changes in the following pattern, and the trained rats did not retain on the rotarod for a longer duration of time. The rats were confused and showed additional time duration to find the retarding place or the marked place after subjection for training. It is one of the ways to identify externally, without the dissection of animals, the neural or mental condition of the rats. As the symptoms of neurodegeneration include the deterioration of neural and motor functions, the actions produced by the body of rats, such as the duration of time spent on the rot rod or the time taken by the animal to identify the marked location, can justify the need for further biochemical tests. The test shows the results as per the reviewed literature. In the rotarod test, the normal saline grouped rats could retain on the rod for 764.2 s, when the rats were injected with the haloperidol the time retained by the rats on the rod decreased to 562.3 s, when the rats were injected with the rosiglitazone and haloperidol the time was increased to the 663.5 s, this indicates that the rosiglitazone can improve the time retained. It is due to the increase in reduction in sugar-induced oxidative stress, which was increased due to the AGE action. The neural cell damage due to continuous oxidative stress, which is caused by the glucose and other sugar molecules in the body, can be suppressed by the action of rosiglitazone. It reduces the NF-κβ and other inflammatory mediators to show beneficial effects in neurodegeneration. It is also indicative of the biochemical test results and the histopathological studies that are performed on the rats [Figure 2].

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Figure 2:

Behavioral parameters, all values are expressed as mean ± SD, when number of rats used in each group (n = 6). Data were subjected to one-way ANOVA followed by Dennett’s test when normal saline was compared to haloperidol control group (disease control), haloperidol and rosiglitazone treated group (Test control) and per se were compared. *a indicates P < 0.0001, *b indicates P < 0.005*** when compared to normal saline. NS: Normal saline, DC: Disease control, TC: Treatment control, P: per se.

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The biochemical tests

It is theoretically being studied that oxidation and inflammation are the root causes of neurodegeneration. The study of the different oxidation parameters in the brain homogenate would provide the extent of the disorder. The values of SOD, GSH and CAT indicate that accordingly. The mean of SOD values for normal control was found to be 2.3 U/mg, 0.0036 nmol/mg and 2.54 mmol/mL, and the haloperidol increased the oxidation levels to 4.34 U/mg, 0.0074 nmol/mg and 1.54 mmol/mL, which was reduced to 3.24 U/mg, 0.0058 nmol/mg and 1.76 mmol/mL with rosiglitazone. In the rats, subjected to the rosiglitazone alone the SOD level was found to be 2.54 U/mg, 0.0048 nmol/mg and 2.20 mmol/mL. The increased levels of SOD, GSH and CAT indicate higher reactions with the oxidant molecules. The drug rosiglitazone acts on the reduction of the free radicals, thereby reducing the oxidant stress and reducing the diseased conditions [Figure 3].

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Figure 3:

The biochemical tests, all values are expressed as mean ± SD, when the number of rats used in each group (n = 6). Data were subjected to one-way ANOVA followed by Dunnett’s test when normal saline was compared to the haloperidol control group (disease control), haloperidol and rosiglitazone treated group (Test control), and per se were compared. *a indicates P < 0.0001, when compared to normal saline. NS: Normal saline, DC: Disease control, TC: Treatment control, P: per se.

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AGE-RAGE parameters

The AGE-RAGE parameters show the increased levels of values in diseased rats, indicating the presence of neurodegeneration. The readings of IL-6, TNF-α and Aβ protein are low, i.e. at the 3 ρg/L mL, 23 ρg/L mL and 0.7 ng/L. The values increased to 10 ρg/L ml, 70 ρg/L mL and 3.5 ng/L; these readings were reduced with the help of rosiglitazone [Figure 4].

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Figure 4:

AGE-RAGE parameter, all values are expressed as mean ± SD, when the number of rats used in each group (n = 6). Data were subjected to one-way ANOVA followed by Dunnett’s test when normal saline was compared to haloperidol control group (disease control), haloperidol and rosiglitazone treated group (Test control) and perse were compared. *a indicates P < 0.0001 when compared to normal saline. NS: Normal saline, DC: Disease control, TC: Treatment control, P: per se, TNF: Tumour necrosis factor, IL: Interleukin.

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Histopathological studies

The cerebrum of the rats was examined under a microscope to study their neural cells, assess damage, and evaluate neurological conditions. This is also required to understand the extent of the treatment after the rosiglitazone ingestion. As it is the comparative parameter, the study also needs the comparison along with the normal saline and per se groups, which would indicate if any changes were to be nullified during the study. The histopathology of the brain tissue of the rats belonging to the normal control group revealed normal cerebral cortex cell bodies and glands. It is due to the group not being treated with any harmful substance. This indicates that no notable abnormalities, such as neuronal degeneration or gliosis (the accumulation of glial cells), were seen in the cerebral cortex tissue. The treatment group serves as a comparative group to assess the effects of rosiglitazone therapy and haloperidol-induced neurotoxicity. The cerebral cortex tissue in this group had significant gliosis, or an accumulation of glial cells, according to the results of the histological study. Significant neuronal deterioration was also noticed. These results suggest that haloperidol administration resulted in significant neuronal death and gliosis. Haloperidol is thus a neurodegenerative agent which acts upon the neural cells and worsens the condition of the disease. The study of cerebral tissue of this group of rats (histopathological study) demonstrates significant gliosis (build-up of glial cells) in the cerebral cortex tissue. This is mild in the condition, as it is the haloperidol and rosiglitazone-treated group that shows significant neuronal deterioration. This shows that the gliosis and neuronal degeneration were reduced due to the rosiglitazone administered along with haloperidol. The cerebral cortex cell bodies in this group’s brain tissue were normal according to the histological analysis. This shows that rosiglitazone injection alone did not result in any appreciable alterations in the cerebral cortex tissue, indicating that it did not result in gliosis or neuronal degeneration. The complete histopathological analysis shows that rosiglitazone is the drug of choice in neural deterioration. Rosiglitazone does not show any action on the neural damage and helps to reduce the damage which is produced by the haloperidol [Figure 5].

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Figure 5:

Histopathology of rat cerebrum, images representing the microscopical characters of the rat’s brain in the Transverse section (TS) and Virtual section (VS), where NS is normal saline, DC is disease control, TC is treatment control, and P is for per se. The tissue were analyzed using haematoxylin & eosin stain, at 10×. TS: Transverse section, VS: Vertical section.

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DISCUSSION

The change in the body weight of the rat is an external parameter that can be a marker to indicate the required action of haloperidol as a prompt toward neurodegeneration.^[42] The effect on dopamine 2 receptor, antagonist action by the haloperidol has a negative effect, which induces neurodegenerative disorder, and rosiglitazone was tested to treat this neurodegeneration.^[43] The effect of rosiglitazone was found to be such that the parameters deciding the fate and extent of neurological disorder were reduced by the rosiglitazone treatment. It is known that the advanced glycation end product has a role in the prognosis of neurodegeneration.^[44]

All these theoretical assessments are true if the biochemical and histopathological changes indicate the same. The test starts with some external and behavioural parameters and leads to biochemical and histopathological studies. The behavioural test marked the memory loss and also the loss of ability to withstand the pressure induced by it. The normal control rats, which were able to withstand a higher degree of pressure or show resistance to a greater extent, were reduced in the diseased rats. The ability of rats was brought to near normal after treatment. Similar action was seen in the biochemical tests where the increased oxidation products such as CAT, SOD and GSH were reduced after treatment with rosiglitazone. Further investigation of the drug’s action on AGE parameters has helped to understand its action on convalescents in this study. The parameters TNF-α, IL-6 and Aβ protein accumulation, indicating the rise in the AGE, were reduced by the rosiglitazone. There are various models through which neurodegeneration has already been studied, and the impact of neurodegeneration through the AGE-RAGE axis and designing an animal model that includes a maximum number of parameters, including oxidation, was a priority of this study. The screening of brain histopathology was designed to understand the impact of cell damage caused by haloperidol and its treatment with rosiglitazone, which inhibits the glycation structures that are expressed with reduced gliosis structure and neural regeneration structures, indicating the impact of rosiglitazone in the treatment of neurodegenerative disorder. The action of the synaptic vesicles and other neural structures is for further studies and needs additional attention. This could further contribute to the changes that are to be considered to understand the impact of PPAR-γ on AGE-RAGE. When compared to a similar study involving haloperidol-induced neurodegeneration, behavioural assessments, including tests such as the Morris water maze and open-field test, highlighted significant cognitive and motor deficits. Treatment with pioglitazone, another PPAR-γ agonist, showed comparable improvements in cognitive and behavioural outcomes, suggesting a shared mechanism of action through PPAR-γ modulation.^[45] Similarly, with the antioxidant studies, comparable pre-clinical studies have shown that PPAR-γ agonists, including rosiglitazone and pioglitazone, enhance antioxidative defences by upregulating the expression of antioxidant enzymes. For instance, studies in Parkinson’s disease models also reported reduced oxidative markers after treatment with these drugs, suggesting a generalisable antioxidative mechanism across neurodegenerative conditions. Similar results were seen in the histopathological studies, and this shows that rosiglitazone can deliver the required action against neurodegeneration.^[46] The study that has to be further taken up or still to be considered as a limitation is the investigation and comparison of other pathways acting as the neurodegenerative disorder treatment with that of the action pathway of rosiglitazone.

CONCLUSION

These results suggest that the blockage of AGE has an effect on the neurodegeneration and the development of the rat model. It is indicative from the pathway discussed that the effect of PPAR-γ blocker drugs can act by reducing the effect produced by the AGE and blocking the RAGE to cure neurological conditions. It was indicative from the results that the disease control has a higher amount of the antioxidant being produced which acts on the neurological cells as said earlier and the per se treated group which was subjected to the drug only showed values equivalent to that of the normal and the treated groups also show that the disease has been caused which is reduced by the drug and the conditions are improvised by the drug. The present study is everlasting since the AGE is associated with the diet and can have several other triggering centres as well, which is not easy to explore. Further study on the extent of impact needs to be considered.