Biochemical Changes in Alloxan-Induced Diabetes Rat Liver and Kidney Dosed Artesunate

Diabetes is a disease marked by elevated blood sugar levels, and the second leading cause of renal diseases and blindness worldwide. Artesunate is an antimalarial drug, that has been reported to have hypoglycemic potential, but to the best of our knowledge, much work has not been done to explore the biochemical and clinical implications of administration of artesunate on a diabetic subject. This study investigates biochemical changes in terms of oxidative status associated with oral administration of artesunate on diabetic animal model. Twenty eight male Wistar rats weighing averagely 200g were divided into four groups of seven rats each, Group A-control, B- Diabetes only, C- Artesunate only, D- Diabetes + Artesunate. Diabetes was induced intraperitoneally, at a single dose of Alloxan(160mg/kg body weight(bw). Artesunate was administered orally in aqueous solution at 2.90mg/kg bw on day one, and at 1.45mg/kg bw on the subsequent 7days. Spectrophotometric technique was used for biochemical analysis in serum, kidney and liver homogenates. Aspartate amino transferase (AST) and Alanine amino transferase (ALT) activities as well as Creatinine concentration were significantly (P<0.05) increased in group B compared with control, while group D showed a significant (P<0.05) decrease compared with group B. Total protein concentration was significantly(P<0.05) increased in group B compared with control, while group D  showed an insignificant  decrease compared  with group B. Moreover, Superoxide dismutase(SOD) and Catalase(CAT) activities as well as  Reduced glutathione (GSH) concentration were significantly(P<0.05) decreased in group B compared with control, and were restored near to the control level significantly (P<0.05) in group D, compared with group B. Malondialdehyde (MDA) concentration was significantly (P<0.05) increased in group B compared with control, but was reversed significantly (P<0.05) in group D compared with group B. Artesunate ameliorates oxidative stress in diabetic rats liver and kidney, possess hypoglycemic effect, hence antioxidant and hypoglycemic effects of artesunate may be beneficial to diabetic liver and kidney. Keywords: Artesunate, Diabetes mellitus, Hypoglycemia, Alloxan, Antioxidant. DOI : 10.7176/ALST/72-03 Publication date :March 31 st 2019


INTRODUCTION
Diabetes mellitus is a leading cause of morbidity and mortality worldwide. It is a metabolic disorder of multiple etiology characterized by chronic hyperglycemia with disturbance of carbohydrate, fat, and protein metabolism which may result from defects in insulin secretion, insulin action or both (Shoback, 2011). Environmental factors, genes and pathological condition like autoimmune eradication in beta cells of the pancrease that leads to deficiency of insulin and some other abnormalities causing insulin resistance are involved in the etiology of diabetes (Okur et al., 2017). Type 1 diabetes (Insulin Dependent Diabetes Mellitus) is caused by a lack of insulin due to the destruction of insulin-producing beta cells in the pancreas. Here, the body's immune system attacks and destroys the beta cells. Type 1diabetes indicates the processes of destruction of beta-cells that may ultimately lead to diabetes mellitus in which insulin is required for survival to prevent the development of ketoacidosis, coma and death. An individual with a type 1diabetes process may be metabolically normal prior to when the disease is clinically manifest, but the process of beta-cell destruction can be detected. Presence of anti-GAD usually characterize type 1 diabetes mellitus, islet cell or insulin antibodies which identify the autoimmune processes that lead to beta-cell destruction (WHO 1999). Beta cells destruction in the pancrease provokes an insulin insufficiency hence, diabetic patients with type 1 are subjected to daily insulin administration to normalize the sugar concentration present in their blood (WHO, 2016) .Type 2 diabetes (Non Insulin dependent diabetes Mellitus), is the most common form of diabetes which is characterized by insulin disorder, action and insulin secretion, either of which may be the predominant feature. Both are usually present at the time that this form of diabetes is clinically manifest (WHO 1999). In type 2 diabetes, individual has relative rather than Aldrich company Louis, U.S.A. and Randox Laboratory limited Ardmore Diamond road, Crumlin Uk. Artesunate (ARTs) (50mg) was purchased from Tuyil Pharmaceutical Company, Ilorin, Kwara state, Nigeria.

Experimental Animal
Twenty eight male Wistar rats weighing averagely 200g were purchased from a reliable source. They were housed in a cage under standard laboratory condition of 12 hours light and 12 hours dark period. Acclimatization to laboratory condition was done for 2weeks before the commencement of the experiment. All the procedures involving the use of laboratory animal were in accordance to the Institute Animal Ethics Committee regulation approved by the committee for the purpose of control and supervision of Experiments on Animals (CPCSEA).

Experimental Design
The rats were randomly divided into four groups of seven rats per group and treated for eight days after 72hours of alloxan induction.. Group A (control): Rats received standard rat pellet and distilled water only ad libitum. Group B (Diabetes only): Rats were induced diabetes with 160mg/kg body weight (bw) Alloxan already dissolved in 0.9% normal saline and fed with standard rat pellet and distilled water ad libitum. Group C (Artesunate only): Rats were dosed Artesunate only at 2.90mg/kg bw on first day and at 1.45mg/kg bw for the subsequent 7days. Group D (Diabetes + Artesunate) : Rats were induced diabetes followed by 2.90mg/kg bw of Artesunate on first day, and 1.45mg/kg for subsequent 7days.

Diabetes Induction
Fourteen Albino rats in group B (Diabetes only) and D (Diab.+Artesunate) were fasted overnight and injected with Alloxan already dissolved in 0.9% normal saline intraperitoneally at a single dose of 160mg/kg body weight (Ashok et al., 2007). The diabetes states of the rats were ascertained by measuring their blood sugar levels after 72 hours using a glucometer.

Administration of Artesunate
The administration of Artesunate commenced 72hours after diabetes induction. Rats in group C (Artesunate only) and group D (Diab.+Artesunate) were administered Artesunate orally at 2.9mg/kg bw on first day and 1.45mg/kg bw on the subsequent 7days (Salman et al., 2017). The dosage was chosen to mimic that of human.

Sample collection and Preparation
Experimental rats were fasted overnight and weighed before sacrifice was done by cervical dislocation, animals were dissected for sample collection.

Collection and preparation of blood serum
The blood samples were carefully collected by cardiac puncture using sterile needle and syringe, drained into sterilized sample bottles and left for 30munites, centrifuged at 3000rpm for 10minutes.The serum was picked to sterilized sample bottles for biochemical analysis.

Collection and preparation of Tissue
The renal and liver tissues of the rats were quickly removed after sacrifice, weighed, washed repeatedly in chilled 1.15M Kcl and placed in clean sample bottle. Renal and hepatic tissues were homogenized with sucrose phosphate buffer using homogenizer.

Determination of Serum Alanine Amino Transferase (ALT) and Aspartate amino Transferases (AST) Activities
The ALT activity was measured by monitoring the concentration of the pyruvatehydrazon formed with 2,4 dinitrophenyhydrazine as specified by Reitman and Frankel 1951. AST activity was determined by monitoring the concentration of oxaloacetate hydrazine formed with 2,4-dinitrophenylhydrazine as specified by assay kit.

Determination of serum Total protein concentration
Serum total protein concentration was determined based on the method described by Folin-lowry (Lowry et al., 1951). Protein reacts with follin-ciocalteau reagent to give a blue coloured complex that absorbed at wavelength of 500nm

Determination of lipid peroxidation
Estimation of lipid peroxidation was based on the reaction of Malondialdehyde (MDA) with Thiobarbituric acid (TBA) forming MDA-TBAR adduct that absorbed strongly at 532nm,as described by Varsney and Kale(1990).

Determination of Superoxide Dismutase (SOD) and Catalase (CAT) activities
Superoxide dismutase (SOD) activity was determined by method of Misra and Fridovich (1972). SOD inhibits auto oxidation of epinephrine to adrenochrome and absorbance read at 480nm. Catalase activity was measured according to the method of Aebi (1983). The rate of decomposition of H 2 O 2 was measured spectrophotometrically from change in absorbance at 240nm.

Determination of Reduced glutathione (GSH) Concentration
Reduced glutathione (GSH) estimation was based on the development of relatively stable yellow colour that absorbed at 412nm when 5,5, dithiobis-2-nitrobenzoic acid is added to sulphydryl compounds as described by Moron et al., 1979.

Determination of serum Creatinine concentration
Serum creatinine level was determined by the method specified in the assay kit. Creatinine in alkaline solution reacts with Picric acid to form a coloured complex that was measured spectrophotometrically. The amount of the complex formed was directly proportional to the creatinine concentration.

Statistical Analysis
All data were presented as Mean ± Standard error of Mean (SEM). Statistical analysis was carried out using Graphpad prism 5 Software. One way Analysis of Variance (ANOVA) was used for the comparison of relative expression levels for different groups, this was followed by Turkey Post hoc Test. Level of significance was taken at (P<0.05) for six rats per group 'a' represents significant decrease when compared with Diabetes only group 'b' represents significant decrease when compared with control

Alanine amino transferase (ALT) activity.
There is a significant (P< 0.05) increase in ALT activity in group B (Diabetes only) when compared with group A (Control) and other groups. An insignificant decrease was observed in group C (Artesunate only) when compared with group A (control). A significant (P< 0.05) decrease was observed in group D (Diab. + Artesunate) when compared with group B (Diabetes only).
Level of significance was taken at (P<0.05) for six rats per group 'a' represents significant increase when compared with control and other groups 'b' represents significant decrease when compared with Diabetes only.

Aspartate amino transferase (AST) activity
Aspartate amino transferase (AST) activity in group B (Diabetes only) was significantly (P< 0.05) increased when compared with control and other groups. An insignificant decrease is AST activity was observed in group C (Artesunate only) when compared with group A (control), while a significant (P<0.05) decrease was observed in group D (Diab.+Artesunate) when compared with group B (Diabetes only). Level of significance was taken at (P<0.05) for six rats per group 'a' represents significant increase in diabetes only group when compared with control and artesunate only group Level of significance was taken at (P<0.05) for six rats per group 'a' represents significant decrease when compared with control and other groups. 'b' represents significant increase when compared with control and diabetes only. 'c' represents significant increase when compared with control and diabetes only group

Reduced glutathione (GSH) concentration
Reduced glutathione (GSH) concentration was significantly (P< 0.05) decreased in group B (Diabetes only) when compared with control and other groups, group C (Artesunate only) showed a significant (P< 0.05) increase in GSH concentration when compared with control, group D (Diab.+Artesunate) also, when compared with group B (Diabetes only) showed a significant (P< 0.05) increase in GSH concentration. Level of significance was taken at (P<0.05) for six rats per group 'a' represents significant decrease when compared with control and other groups. 'b' represents significant increase when compared with control and diabetes only 'c' represents significant increase when compared with control and diabetes only group

Catalase (CAT) and Superoxide dismutase (SOD) activities in the Liver
Catalase (CAT) (A) and Superoxide dismutase (SOD) (B) activities were significantly (P< 0.05) decreased in group B (Diabetes only) when compared with control, group C (Artesunate only) showed an insignificant increase in activities of SOD and Catalase compared with control, while a significant (P< 0.05) increase was observed in group D (Diab.+Artesunate) when compared with group B (Diabetes only).

(A)
Level of significance was taken at (P<0.05) for six rats per group 'a' represents significant decrease when compared with control and other groups. 'b' represents significant increase when compared with Diabetes only (B) Level of significance was taken at (P<0.05) for six rats per group 'a' represents significant decrease when compared with control and other groups. 'b' represents significant increase when compared with Diabetes only

DISCUSSION
Glycemic homeostasis refers to glucose balance or control within circulation in living organisms. It is normally and largely compromised in diabetes state. When the compromise is worsen, it leads to several complications including retinopathy, nephropathy and neuropathy which are all collectively referred to as diabetic complications and these are the principal factors in morbidity and mortality often associated with diabetes (Ighodaro et al., 2018).Alloxan and streptozotozin are the most popular diabetogenic agents used for assessing the antidiabetic or hypoglycemic capacity of test compounds, Notably, alloxan is far less expensive and much more readily available than streptozotozin (Ighodaro et al., 2018). The Cytotoxic action of Alloxan as a diabetogenic agent is mediated by Reactive Oxygen Species(ROS) (Szkudelski 2001), Alloxan, a toxic glucose analogue accumulates in pancreatic beta cells via the action of glucose transporter referred to as GLUT2 (Lenzen, 2008) . In the presence of intracellular thiol like glutathione, Alloxan generates ROS in a cyclic redox reaction with its reduction product called dialuric acid. The process of autoxidation of dialuric acid leads to generation of superoxide radicals, hydrogen Peroxide and finally, hydroxyl radicals. These generated hydroxyl radicals are ultimately responsible for the death of beta cells which have low antioxidative defense capacity resulting in the state of Insulin-dependent alloxan diabetes (Lenzen, 2008). In figure 3.1, Blood sugar level after 72hours of Alloxan induction before artesunate administration showed a significant (P <0.05) increase in Group B (Diabetes only) and Group D (Diab. + Artes.) when compared with group A (control) and group C (artesunate only), this showed that, beta cell destruction in pancrease has been occasioned by the action of reactive oxygen species generated by alloxan metabolic intermediates. Moreover, after the treatment, group D (Diab.+artes.) showed a significant (P<0.05) decrease in blood sugar concentration restored near to the control comparably with group B (Diabetes only). This suggests hypoglycemic potential of artesunate and its possible antioxidant ability.
The aminotransferases are the most frequently utilized and specific indicators of hepatocellular necrosis. These enzymes, aspartate aminotransferase (AST) and alanine amino transferase (ALT) catalyze the transfer of the α-amino acids of aspartate and alanine respectively to the keto-group of α-ketoglutaric acid. Serum, AST and ALT are the most sensitive biomarkers used in the diagnosis of liver diseases (Pari and Murugan, 2004). During hepatocellular damage, varieties of enzymes normally located in the cytosol are released into the blood flow. Their quantification in plasma is useful biomarkers of the extent and type of hepatocellular damage (Pari and Murugan, 2004).Serum ALT and AST are liver enzymes,that leak out into general circulation when there is damage to the liver cells. Both AST and ALT have been reported as useful predictors of liver pathology (Anderson et al., 2000). In figure 3.2 and 3.3 ALT and AST activities were significantly (P< 0.05) increased in group B (Diabetes only) when compared with control and other groups. There was an insignificant decrease in AST and ALT activities in group C (Artesunate only) when compared with group A (control), administration of artesunate in group D (Diab.+Artesunate) occasioned a significant (P< 0.05) decrease in serum activities of these enzymes compared to group B (Diabetes only). Artesunate or its metabolites might have exhibited hepatoprotective effects that led to decrease in serum ALT and AST activities.
Proteins are constituents of muscle, enzyme, hormone, and other key functional and structural entities in the body. Elevated levels of protein are found in dehydration while reductions are seen mainly in malnutrition, impaired protein synthesis, protein losses, and excessive protein breakdown (Doumas and Bayse, 1981). In figure 3.4, total protein concentration was significantly (P< 0.05) increased in group B (Diabetes only) when compared with control. Group D (Diab.+Artesunate) showed an insignificant decrease in serum total protein when compared with group B (Diabetes only), while comparison between group C (Artesunate only) and control was not statistically significant. Dehydration that occurred as a result of diabetes complication might have resulted in elevated level of serum total protein in group B (Diabetes only) while slight reduction in serum total protein might be traced to effect of artesunate or its metabolite.
Serum creatinine is an important indicator of renal function. It is synthesized in the kidney from amino acids Arginine, glycine and methionine. An elevated blood creatinine level is observed with marked damaged to functioning nephrons (Gross et al., 2005). In figure 3.5, alloxan occasioned a significant (P< 0.05) increase in serum creatinine in group B (Diabetes only) when compared with control. There was an insignificant decrease in group C (Artesunate only) compared with control, while group D (Diab.+Artesunate) showed significant (P< 0.05) decrease in creatinine concentration when compared with group B (Diabetes only). Interestingly, this is consistent with the work of Chiagoziem et al., (2012). This revealed that, artesunate might possess properties that ameliorate oxidative stress in the Kidney.
The constituents of cell biomembranes are liable to free radical and active oxygen damage. The actions of these free radicals trigger cell damage by covalently binding with cellular macromolecules and formation of lipid peroxidation (Ajay et al., 2010) which are implicated in the processes such as carcinogenesis, inflammation and aging (Ames et al., 1993).Free radicals produced from oxidative stress have potential to react quickly with lipid in the body to form lipid peroxide (LPO) (Rukkumani et al., 2004). Result obtained in figure 3.6 showed a significant (P< 0.05) increase in MDA concentration in group B (Diabetes only) when compared with control and other groups. Group C (Artesunate only) showed an insignificant increase in MDA concentration when compared with control while group D (Diab.+Artesunate) showed significant decrease when compared with group B (Diabetes only). This reflects that, artesunate administration "in diabetes state" may stabilize or protects biomembranes in renal tissue.
Superoxide dismutase (SOD) and Catalases (CAT) are enzymatic antioxidants. SODs are class of closely related enzymes catalyzing the breakdown of superoxide anion to oxygen and Hydrogen peroxide (Zelko et al., 2002). They are present in almost all aerobic cells and in extracellular fluid (Johnson et al., 2005). Catalase enzyme catalyzes conversion of hydrogen peroxide to water and oxygen using iron or Manganese co factor. Tissues are protected from oxidative stress with increase catalase activity while on reduction are under attack of oxidative stress (Chelikani et al., 2004). In figures 3.7(A and B), SOD and Catalase activities were significantly (P< 0.05) decreased in group B (Diabetes only) when compared with control. Moreover, a significant (P<0.05) increase was observed in group C (Artesunate only) when compared with control and group B (diabetes only), furthermore, a significant (P< 0.05) increase was observed in group D (Diab.+Artesunate) when compared with group B (Diabetes only). Also, in figure 3.9 (A and B), liver catalase and superoxide dismutase activities were significantly (P<0.05) decreased in group B (Diabetes only) when compared with control and other groups, Advances in Life Science and Technology www.iiste.org ISSN 2224-7181 (Paper) ISSN 2225-062X (Online) DOI: 10.7176/ALST Vol.72, 2019 33 while these enzymes activities were restored significantly (P<0.05) in group D (Diab.+ Artesunate) when compared with group B (Diabetes only). An insignificant increase was observed in group C (Artesunate only) when compared with group (A) control. Artesunate or its metabolites might have elicited pathways that led to elevation of these antioxidant enzymes in renal and hepatic tissues.
Reduced glutathione (GSH) serves as non-enzymatic antioxidant biological molecule in the body tissue. It functions to remove non radical compounds like hydrogen peroxide, Alkoxyl radical, and superoxide anion from tissue (Townsend et al., 2003). In figure 3.8, alloxan occasioned a significant (P< 0.05) decreased in GSH concentration in group B (Diabetes only) when compared with control and other groups, Group C (Artesunate only), showed a significant (P<0.05) increase in GSH concentration when compared with control. Moreover, group D (Diab.+Artesunate) showed significant (P<0.05) increase when compared with group B (Diabetes only). Artesunate might have elicited pathways that enhances reduced glutathione concentration in renal tissue. Conclusion: from the result of this present work, artesunate ameliorates oxidative stress in diabetic rats Liver and Kidney hence, antioxidant effect of artesunate may be beneficial to diabetic Kidney and liver. Moreover, diabetic patients that have malaria may opt for artesunate to benefit themselves for its antioxidant and hypoglycemic potential.