The neuroprotective effect of simvastatin on the cerebellum of experimentally-induced diabetic rats through klotho upregulation: An immunohistochemical study

https://doi.org/10.1016/j.jchemneu.2020.101803Get rights and content

Highlights

Abstract

Background

Diabetes mellitus is a multifactorial metabolic disorder that is complicated by multi-organ dysfunction including CNS. Klotho is an anti-aging protein expressed in Purkinje cells of the cerebellum. Klotho protects against the development of several neurodegenerative diseases. Simvastatin is a lipophilic statin that can enhance klotho expression.

Aim of the study

This study was designed to investigate cerebellar structural changes in diabetes, klotho expression in the cerebellum of diabetic rats and the neuroprotective effect of simvastatin.

Materials & methods

24 adult albino rats were divided into 4 groups; control, simvastatin, diabetic (induced by single intraperitoneal injection of STZ 45 mg/kg) and diabetic treated by simvastatin (10 mg/kg once daily) after confirmation of diabetes. Rotarod test was performed for evaluation of motor coordination. Blood glucose and insulin levels were estimated for confirmation of diabetes. Reduced glutathione (GSH) and malonaldehyde (MDA) in cerebellar tissues were evaluated. The cerebellar samples were prepared for histological and immunohistochemical staining.

Results

The latency time on the rotarod was reduced in diabetic rats. Cerebellar structure was disturbed in diabetic group. Oxidative stress was evidenced by increased MDA and reduced GSH. Klotho expression was downregulated and caspase-3 was increased in diabetes. Simvastatin increased the latency time. Simvastatin diminished the changes in oxidative stress markers and succeeded to ameliorate the diabetic induced cerebellar changes. Simvastatin enhanced Klotho and diminished Caspase-3 expression.

Conclusion

Simvastatin can ameliorate diabetic induced cerebellar changes through minimizing oxidative stress, enhancement of Klotho expression and reduction of apoptosis.

Introduction

Diabetes mellitus is a common multifactorial endocrinal metabolic disease that is characterized by alternated carbohydrates, lipids and protein metabolism. Glucose metabolism is disturbed due to deficiency in insulin secretion, insulin action, or both leading to hyperglycemia (Lebed et al., 2008). Long term hyperglycemia causes microvascular and macrovascular complications (Zimmerman, 2016) in different organs including kidney, retina, and brain (Özdemir et al., 2016; Prabhakar, 2016). Diabetes mellitus is accompanied by behavioral abnormalities and reduced motor activity (Kumar et al., 2010). Oxidative stress is one of the mechanisms of hyperglycemia-induced complications (Asmat et al., 2016; Chawla et al., 2016; Yaribeygi et al., 2019).

Several studies proved that different parts of the CNS including; cerebral cortex (Romano et al., 2018), hippocampus (Hao et al., 2019) and cerebellum (Niyomchan et al., 2019) are affected in diabetic rats. The cerebellum has a well-established role in controlling motor functions such as posture, balance, coordination, and skilled learning (Beckinghausen and Sillitoe, 2019). Also, there is mounting evidence that it can play a critical role in non-motor functions such as cognition, emotion, and language (Beckinghausen and Sillitoe, 2019). The cerebellar structural changes induced by diabetes are not co-related to the glycemic control or the disease duration (Lunetta et al., 1994).

Klotho, an anti-aging protein, was discovered by Kuro-o and coworkers by insertional mutations in mice in 1997 (Kuro-o et al., 1997). Mice with knocked-out klotho gene developed premature aging, atherosclerosis, osteopenia, organ atrophy, neuronal degeneration and parkinsonian like gait abnormalities with reduction of the number of Purkinje cells in the cerebellum (Kuro-o et al., 1997; Torbus-Paluszczak et al., 2018). Though, klotho is predominantly produced by the kidney, it is, also, expressed in large amounts in the brain by ependymal cells of the choroid plexus and Purkinje cells of the cerebellum (Olauson et al., 2017). In Purkinje cell, Klotho is present in the cell body, dendrites and axon and it is most abundant near the nuclear membrane (Boksha et al., 2017). Recently, the neural Klotho is evidenced to protect against the development of several neurodegenerative disorders such as Alzheimer's disease and multiple sclerosis (Aleagha et al., 2015; Porter et al., 2019). It is thought that one of its neuroprotective mechanisms is related to its ability to alleviate oxidative stress (Abraham et al., 2016; Boksha et al., 2017). So, the enhancement of Klotho expression improves neuronal health and function (Vo et al., 2018).

Simvastatin is a statin drug that inhibits hydroxymethylglutaryl coenzyme A reductase enzyme (Bedi et al., 2016). It is a lipophilic statin that can cross the blood brain barrier (Tramontina et al., 2011). Beside its hypocholesterolemic effect, it has other pleiotropic effects, including, antineoplastic, antioxidant, anti-apoptotic and anti-inflammatory (Lacerda et al., 2014; Al-Rasheed et al., 2018; Liang et al., 2019; Adejumo et al., 2019). Recently, it was reported that simvastatin had a neuroprotective effect via enhancement of klotho expression (Adeli et al., 2017).

To the best of our knowledge, neither the klotho expression in the cerebellum nor the neuroprotective effect of simvastatin on it has been evaluated before in diabetic rats. In the present study, we aimed to investigate cerebellar structural changes in diabetes, klotho expression in the cerebellum of diabetic rats and the neuroprotective effect of simvastatin using histopathological and immunohistochemical procedures.

Section snippets

Experimental Animals

Twenty four adult male albino rats, weighing 200 – 250 g were obtained from Mansoura Experimental Research Center, Faculty of Medicine, Mansoura University, Egypt. They were kept under controlled condition of temperature (23 ± 3 °C), and relative humidity throughout acclimatization and experimental periods, and fixed 12:12 -hs light/dark cycle. They were allowed free access to food and water.

Experimental design and treatments

Rats were randomly divided into four groups (six rats in each group) control, simvastatin, diabetic, and

Effect of simvastatin on motor activity (rotarod test)

The latency time was significantly reduced in diabetic rats in comparison with the control group. Simvastatin treatment of diabetic rats significantly increased the latency time as compared with the diabetic group. Simvastatin group showed no significant difference versus the control group (Fig. 1).

Effect of simvastatin on body weight and cerebellar weight

After 8 weeks of STZ injection, the final body weight and the weight of the cerebellum of the diabetic rats showed a significant decrease versus the control group. Simvastatin treatment of diabetic

Discussion

In the present work, the diabetic rats showed criteria of diabetes; increased blood glucose level, decreased serum insulin level and decreased body weight in agreement with previous studies (Choi et al., 2009; Furman, 2015; Al-Rasheed et al., 2017).

Increased blood glucose level causes an elevation in the brain glucose (Rom et al., 2019). The increased intracellular glucose is neurotoxic (Xie et al., 2018) as auto-oxidation of high intracellular glucose leads to the production of reactive oxygen

Author statment

The study was designed by Samira Lotfy Abd-Elhady. Ola Mohammed Youssef, Amira Ibrahim Morsy, Mona A. El-Shahat, and Amany M Shams collected and analyzed the data. Ola Mohammed Youssef, and Mona A. El-Shahat prepared the manuscript. All the authors approved the final version of the manuscript.

Ethical statment

All the experiments were carried out according to the regulations and rules laid down by the committee of animals’ experimentation of Mansoura University. The study was approved by Institutional Review Board of Mansoura faculty of medicine (MD.18.11.103).

Declaration of Competing Interest

There is no conflict of interest

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