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UK Journal of Pharmaceutical and Biosciences Vol. 3(1), 01-11, 2015 RESEARCH ARTICLE

Modulatory Effect of Lovastatin on Therapeutic Efficiency of Conventional

Antidiabetics on Pancreatic and Cardiovascular Complications of Diabetes

Ehab A.M. EL-Shoura

1

, Basim A.S. Messiha

2*

, Amira M. ABO-Youssef

2

, Ramadan A.M. Hemeida

1

1Department of Pharmacology, Faculty of Pharmacy, Azhar University, Assuit, Egypt. 2Department of Pharmacology, Faculty of Pharmacy, Beni Sueif University, Beni Sueif, Egypt.

Article Information Received 10 October 2014

Received in revised form 24 Feb 2015 Accepted 25 Feb 2015

Abstract

The aim of the present study is to assess the possible modulatory effect of lovastatin (LOVA) on

metformin (MET) and gliclazide (GLIC) on pancreatic and cardiovascular complications in rats

with experimentally induced diabetes. Male Swiss rats were randomly assigned to seven groups.

Group 1 served as normal control group. Group 2 served as diabetic control group, treated with

streptozotocin (STZ) alone. Groups 3, 4, 5, 6 and 7 received LOVA, MET, GLIC, LOVA plus MET

and LOVA plus GLIC, respectively, for 30 consecutive days after treatment with STZ. To evaluate

the modulatory effect of LOVA, blood glucose level was assessed coupled with pancreatic tissue

levels of oxidative stress markers including thiobarbituric acid reactive substances (TBARS)

production, glutathione reduced (GSH) stores, total nitrite (NO2

-) production as well as

glutathione-S-transferase (GST), superoxide dismutase (SOD) and catalase (CAT) activities.

Degree of lipid derangement was evaluated by measuring serum levels of

3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, triglycerides (TGs), total cholesterol (TC), low

density lipoprotein-cholesterol (LDL-C) and high density lipoprotein-cholesterol (HDL-C). Serum

levels of C-reactive protein (CRP) and interleukin-6 (IL-6), as well as blood fibrinogen (FBG) level

and white blood cells (WBCs) count were evaluated to assess generalized inflammatory and

hematological disorders. Combination of LOVA with either MET or GLIC significantly corrected

STZ-induced alterations of serum levels of glucose, HMG-CoA reductase, TGs, TC, LDL-C,

HDL-C, CRP and IL-6, tissue contents of TBARS, GSH, NO2-, GST and SOD, as well as blood FBG

level and WBCs count as compared to either drug alone. LOVA combination with MET but not

with GLIC could modulate serum HDL-C level, whilst LOVA combination with GLIC but not with

MET could modulate pancreatic tissue CAT activity. These results show that combination of

LOVA with MET or GLIC significantly modulates their effects on diabetes-induced pancreatic and

cardiovascular complications, including hyperglycemia, oxidative and nitrosative stress,

dyslipidemia, inflammatory disorders, hyperfibrinogenemia and leucocytosis.

Keywords:

Lovastatin, Metformin, Gliclazide, Diabetes, Vascular disease.

*

Corresponding Author:

E-mail: drbasimanwar2006@yahoo.com Tel.: +201224459164

1 Introduction

Diabetes, a serious metabolic disorder of carbohydrates, fats and

proteins, affects a large number of populations in the world. Diabetes

is either pancreatic or extra-pancreatic in origin and causes many

complications on nearly all body tissues, including cardiovascular

system, kidney, liver and others. Microvascular complications are

behind a massive percentage of diabetes morbidity and mortality

cases., including retinopathy, neuropathy and nephropathy, and

macrovascular complications, including heart attack, stroke and

peripheral vascular disease, with cardiovascular disease (CVD)

being the main cause of mortality in diabetic patients1.

The 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase

inhibitor, lovastatin (LOVA), is a well-known inhibitor of cholesterol

synthesis and, along with second generation statins, is considered

the first line treatment for hypercholesterolemia. Since dyslipidemia

mediates many diabetic complications, specially, cardiovascular and

UK Journal of Pharmaceutical and Biosciences

Available at www.ukjpb.com

(2)

Messiha et al. Modulatory Effect of Lovastatin on Therapeutic Efficiency

UK J Pharm & Biosci, 2015: 3(1); 2 hematological complications, statins may be of great value in

reducing such complications2. Statins have also been

demonstrated to exert anti-inflammatory effects3.

The standard antidiabetic biguanide drug metformin (MET) is widely

popular. It acts as a cellular AMP-activated protein kinase activator,

a well-known cellular metabolic sensor. The antihyperglycemic

properties of metformin are mainly attributed to its suppression of

hepatic glucose production, especially hepatic gluconeogenesis, and

slightly increased peripheral tissue insulin sensitivity4. MET,

independent of its antidiabetic effect, was reported to offer protection

against vascular and other inflammatory complications5.

The second generation sulfonylurea agent, gliclazide (GLIC), is a

common antidiabetic acting by stimulating pancreatic insulin release

through binding to specific ATP-sensitive potassium channels6. It

was reported to possess good antioxidant activity not related to its

antidiabetic potential7.

Based on the facts listed above, the aim of the present study was to

assess the possible modulatory effect of LOVA on therapeutic

efficiency of MET and GLIC against experimental streptozotocin

(STZ)-induced diabetes in rats concerning pancreatic and vascular

complications. For fulfillment of this purpose, serum level of glucose

and pancreatic tissue levels of thiobarbituric acid reactive

substances (TBARS), glutathione reduced (GSH), total (NO2-),

glutathione-S-transferase (GST), superoxide dismutase (SOD) and

catalase (CAT) were measured. Degree of lipid derangement was

evaluated by measuring serum levels of HMG-CoA reductase,

triglycerides (TGs), total cholesterol (TC), low density

lipoprotein-cholesterol (LDL-C) and high density lipoprotein-lipoprotein-cholesterol (HDL-C).

To assess inflammatory and hematological abnormalities, serum

C-reactive protein (CRP) and interleukin-6 (IL-6) levels, in addition to

white blood cells (WBCs) count were measured.

2 Materials and Method

2.1 Animals

Adult male swiss rats (150-180 g) were procured from central animal

house, Faculty of Medicine, Assiut University (Assiut, Egypt). The

animals were acclimatized to the animal room condition for at least a

week at 25 ± 2 0C with 12/12-hour light/dark cycles prior to the

experiment. All animals were supplied with commercial pellet food

and water ad libitum. All animal housing and handling were

conducted according to the recommendations of the National

Institutes of Health Guide for Care and Use of Laboratory Animals

(Publication No. 85-23).

2.2 Drugs and Chemicals

Test agents LOVA, GLIC and STZ as well as thiobarbituric acid

(TBA), 5,5`-dithio-bis-(2-nitrobenzoic acid) (DTNB), N-(1-Naphthyl)

ethylenediamine dihydrochloride (NEDD) and glutathione reduced

(GSH) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA)

and protected from sunlight. MET was obtained from Chemical

Industries Development CO. (CID; Giza, Egypt). HMG-CoA

reductase ELISA kit was obtained from Wuhan EI-Aab Science Co.

(St. Biopark, Wuhan, China). C-reactive protein (CRP) was obtained

from Assaypro LLC (St. Charles, MO, USA). Serum glucose kit, erum

triglyceride (TG), total cholesterol (TC) and high density

lipoprotein-cholesterol (HDL-C) kits were obtained from Biodiagnostic Co.

(Cairo, Egypt). Serum interlukin-6 (IL-6) kit was obtained from Glory

Science Co. (St, Del Rio, USA). Analytical grade was a necessity in

all other chemicals and solvents.

2.3 Experimental design

Male Swiss rats were randomly assigned to seven groups, each of 6

to 7 rats. Group 1 served as normal control group, receiving only

vehicles. Group 2 received a single i.p. dose of streptozotocin (STZ;

50 mg/kg)8 dissolved in cold citrate buffer (0.1 M, pH = 4.5) and

served as diabetic control. Groups 3, 4, 5, 6 and 7 received LOVA

(15 mg/kg/day)9, MET (100 mg/kg/day)10, GLIC (20 mg/kg/day)11,

LOVA plus MET and LOVA plus GLIC, respectively, by oral gavages

for 30 consecutive days starting on 4th day after treatment with STZ.

2.4 Methodology

2.4.1. Induction of experimental diabetes

Experimental diabetes was induced in 12-hour fasted rats by single

i.p. injection of STZ (50 mg/kg). To prevent initial hypoglycemic effect

of STZ, animals were given 20% glucose solution for the first 24

hours. Induction of diabetes was confirmed 72 hours after STZ

injection through assessment of tail vein blood glucose, after 16

hours of fasting, using a portable glucometer (Accu-Chek, Roche,

Germany). Only animals having fasting blood glucose exceeding 250

mg/dl, coupled with diabetic symptoms like polyphagia,

polydipsiapolyuria, and weight loss were considered diabetic and

included in the study.

After 30 consecutive days of daily drug treatments, rats were lightly

anaesthetized with diethyl ether and sacrificed by cervical

decapitation. Blood samples were collected in tubes containing

EDTA to determine fibrinogen (FBG) and white blood cell (WBCs)

count. Serum was obtained after centrifugation at 3000 x g for 20

minutes using a centrifuge (Beckman GS-6 centrifuge, USA), then

quickly stored at -80oC and used for assessment of serum levels of

triglycerides (TGs), total cholesterol (TC), low density

lipoprotein-cholesterol (LDL-C), high density lipoprotein-lipoprotein-cholesterol (HDL-C),

C-reactive protein (CRP) and interleukin-6 (IL-6). Pancreas was

excised immediately, washed with chilled isotonic saline and stored

(3)

Messiha et al. Modulatory Effect of Lovastatin on Therapeutic Efficiency

UK J Pharm & Biosci, 2015: 3(1); 3 histopathology. Tissue homogenates (10% w/v) were prepared in ice

cold conditions in a homogenizer (Cole-Parmer instrument, USA)

using glass tube and teflon pestle, in phosphate buffer solution. The

homogenate was centrifuged at 1000 g for 10 minutes in refrigerated

centrifuge and the supernatants obtained were used for assessment

of pancreatic tissue levels of thiobarbituric acid reactive substance

(TBARS), glutathione reduced (GSH), total nitrite (NO2-),

glutathione-S-transferase (GST), superoxide dismutase (SOD) and

catalase (CAT). Autopsy samples were taken from pancreas of rats in different groups and fixed in 10% formalin solution in saline to be

used for preparation of liver sections for histopathological study.

2.4.2 Estimation of Biochemical parameters

Serum glucose was estimated by the method of Trinder12. TBARS

eas measured according to the method described by Mihara and

uchiyama13. GSH content was measured by using the method of

Beuter14. GST activity was measured by the method of Habig15. Total

nitrite (NO2-) was done by the method of Montgomery and

Dymock16. Antioxidant enzymes SOD and CAT were measured by

the method of Marklund17 and Abie18, respectively. Serum HMG-CoA

reductase activity and CRP level were assessed according to kit

manufacturer instructions. Estimation of serum TGs was done

according to the method of Fassati and Prencipe19. Estimation of

serum TC was done as previously described20. Estimation of serum

LDL-C was done by using the formula of Friedewald et al, stating that LDL-C = [TC – HDL-C – TG/5]21

. Estimation of serum HDL-C,

was done according to the method of Burstein22. Serum IL-6 was

measured by immunoassay as described earlier23. Blood FBG level

was measured the method of Goodwin24. Estimation of white blood

cells (WBCs) count was done by the method of Berkson25. Tissue

manipulation for preparation of slides for histopathology was

performed according to the method described by Bancroft and

Steven26.

2.5 Statistical analysis

Results are presented as mean ± standard error of mean (SEM). All

the statistical analyses were performed using one way analysis of variance (ANOVA) with turkey’s post hoc comparison test applied

across treatment groups. Significance was based on p value < 0.05.

Data analysis was accomplished using the computer software

program statistical package for social sciences (SPSS; version 20).

3 Results

Administration of STZ to experimental rats resulted in significant

elevations of serum levels of glucose, HMG-CoA reductase, TGs,

TC, LDL-C, CRP and IL-6, pancreatic levels of, blood level of FBG

and blood count of WBCs, as compared with normal rats.

Conversely, STZ administration significantly reduced serum level of

HDL-C and pancreatic levels of GSH, GST, SOD and CAT, as

compared with normal rats.

Treatment of STZ-subjected rats with LOVA, MET, GLIC, LOVA plus

MET or LOVA plus GLIC significantly corrected parameters related

to pancreatic damage, including serum glucose level and pancreatic

tissue levels as compared to STZ control values. Combination of

LOVA with MET or GLIC resulted in significantly better restorations

of serum glucose and pancreatic tissue TBARS, GSH, NO2-, GST

and SOD levels as compared with MET or GLIC monotherapies.

Pancreatic tissue CAT level was significantly corrected only in LOVA

plus CLIC group as compared to STZ control group (Table 1).

Serum markers of dyslipidemia, namely HMG-CoA reductase, TGs,

TC, LDL-C and HDL-C, were significantly corrected by LOVA, MET,

GLIC, LOVA plus MET or LOVA plus GLIC treatments when

compared with STZ control rats. HMG-CoA reductase activity

returned back to normal levels with MET, LOVA plus MET and LOVA

plus GLIC treatments. HDL-C level was increased above normal

control level, except for the GLIC group which was not significantly

different from normal control group. Addition of LOVA to MET

significantly corrected levels of TGs, TC, LDL-C and HDL-C to

degrees better than LOVA or MET monotherapy. Moreover, addition

of LOVA to GLIC significantly corrected TGs and LDL-C levels as

compared to either drug alone (Table 2).

Administration of LOVA, MET, GLIC, LOVA plus MET or LOVA plus

GLIC to rats injected with STZ significantly corrected serum levels of

CRP and IL-6, in addition to blood FBG level and WBCs count as

compared to rats injected with STZ alone. LOVA plus GLIC group

showed blood FBG level not significantly different from that of normal

control group. Combination of LOVA with MET significantly corrected

CRP, IL-6, FBG and WBCs count values to degrees significantly

better than LOVA or MET monotherapies. Similarly, combined effect

of LOVA and GLIC on CRP, IL-6, FBG and WBCs count was

significantly different from individual effects of LOVA and GLIC

monotherapies (Table 3).

Results of histopathological study showed that STZ administration

caused atrophy of islets of Langerhans in addition to perivascular

edema and inflammatory infiltration. Treatment with either LOVA,

MET or GLIC alone corrected such histopathological features

partially, but combination of LOVA with MET or GLIC normalized

pancreatic histological architecture (Fig. 1-7).

4 Discussion

In the present investigation, the modulatory effect of LOVA, a

well-known statin used for treatment of hyperlipidemia, on the beneficial

effects of traditional antidiabetics, namely the biguanide drug MET

(4)

Messiha et al. Modulatory Effect of Lovastatin on Therapeutic Efficiency

UK J Pharm & Biosci, 2015: 3(1); 4 rats with experimental STZ-induced diabetes mellitus (DM) regarding

pancreatic and cardiovascular complications. Results of the present

work showed that a single i.p. dose of STZ in rats resulted in

pancreatic injury evidenced by elevation of blood glucose level

coupled with oxidative injury of pancreatic tissue represented as

elevated TBARS and NO2- levels and suppressed GSH, GST, SOD

and CAT levels (Table 1). STZ-induced diabetes was coupled with

dyslipidemia as evidenced by derangement HMG-CoA reductase,

TGs, TC, LDL-C and HDL-C levels (Table 2). STZ-induced diabetes

was coupled with inflammatory and hematological disorders

represented as elevations of serum CRP and IL-6 levels in addition

to elevations of blood FBG level and WBCs count (Table 3).

Histopathological study strongly supported these biochemical

estimations (Fig. 1-2).

Table 1: Effect of LOVA, MET, GLIC and their combinations on serum glucose level and pancreatic contents of TBARS, GSH, NO2

-,

GST, SOD AND CAT in STZ-treated rats

Treatment Regimen

Parameter

STZ 7+ (LOVA plus GLIC STZ6+ (LOVA

plus MET) STZ5 +

GLIC STZ4 +

MET STZ3 +

LOVA STZ2 Control1 127.26a,b ± 1.605 157.33a,b ± 1.584 148.90a,b ± 1.497 177.83a,b ± 1.514 258.66a,b ± 1.229 301.45a ± 1.829 95.58 ± 2.355 Serum Glucose (mg/dl) 6.66b ± 0.154 8.85a,b ± 0.067 10.50a,b ± 0.169 14.95a,b ± 0.152 16.08a,b ± 0.299 21.25a ± 0.305 5.90 ± 0.051 TBARS content (µmol/g) 4.83a,b ± 0.042 4.01a,b ± 0.047 3.50a,b ± 0.036 2.85a,b ± 0.042 2.30a,b ± 0.019 1.53a ± 0.024 4.43 ± 0.041 GSH content (µmol/g) 2.44b ± 0.028 2.03a,b ± 0.0333 1.73a,b ± 0.042 1.49a,b ± 0.032 1.21a,b ± 0.030 0.54a ± 0.017 2.52 ± 0.019 GST activity (µmol/g) 25.50a,b ± 0.806 31.66a,b ± 0.333 27.83a,b ± 0.600 35.33a,b ± 0.333 39.66a,b ± 0.557 49.50a ± 0.718 18.00 ± 0.683 Total NO2- content

(µmol/g) 3.98b ± 0.054 3.59a,b ± 0.049 2.98a,b ± 0.040 2.56a,b ± 0.065 2.24a,b ± 0.033 0.91a ± 0.046 4.24 ± 0.027 SOD activity (U/g)

0.0151a,b ± 0.00033 0.0145a ± 0.00026 0.0142 a

± 0.00017 0.0145 a

± 0.00022 0.0145 a

± 0.00022 0.0143a ± 0.00012 0.0217 ± 0.00021 CAT activity (U/g)

Data are presented as mean of 6-7 rats ± SEM, 1

Control group, received only vehicles., STZ control group, administered a single i.p. dose of STZ (50 mg/kg), 3

LOVA group, administered LOVA (15 mg/kg/day, p.o.) daily for 30 days after STZ, 4MET group, administered MET (100 mg/kg/day, p.o.) daily for 30 days after STZ, 5GLIC group, administered GLIC (20 mg/kg/day, p.o.) daily for 30 days after STZ, 6LOVA plus MET group, administered LOVA and MET daily for 30 days after STZ, 7LOVA plus GLIC group, administered LOVA and GLIC daily for 30 days after STZ, Statistical analysis was performed by using one way analysis of variance (ANOVA) with turkey’s post hoc comparison test, a

Significantly different from normal control group at p < 0.05, bSignificantly different from STZ control group at p < 0.05.

The diabetogenic agent STZ is a glucopyranose derivative of

l-methyl-l-nitrosourea, and the existence of 2-deoxy-d-glucose in its

structure expedites the preferential uptake of STZ through Glucose

transporter 2 (GLUT2) into the pancreatic β-cells27. In a previous

work, the diabetogenic action of STZ has been explained to cause

alkylation of DNA together with production of nitric oxide and free

radicals, leading to pancreatic damage and decreased insulin

biosynthesis28. This is in harmony with our work results showing

hyperglycemia coupled with oxidative damage of pancreatic tissue

caused by STZ administration.

Dyslipidemia is common in patients with DM and is considered as a

risk factor for the progression of other cardiovascular disease (CVD)

like atherosclerosis29. Generally, lipoprotein abnormalities, presented

as increased plasma levels of very low density lipoproteins (VLDL),

LDL and TGs, are common in diabetics. In agreement with our

results, previous investigators showed similar dyslipidemia in rats

with experimental diabetes30. Deficiency of insulin in diabetic rats is

believed to be associated with increased HMG-CoA reductase

activity, mediating many steps in the process of dyslipidemia31.

Serum level of CRP appears to predict future CVD events, among

(5)

Messiha et al. Modulatory Effect of Lovastatin on Therapeutic Efficiency

UK J Pharm & Biosci, 2015: 3(1); 5 and perivascular disease32. Release of proinflammatory cytokines as

IL-6 is considered a key feature in the process of atherogenesis in

patients with DM33. Additionally, the increased plasma concentrations

of IL-6 is associated with the development of insulin resistance and

type 2 DM, since these cytokines are potential to suppress the action

of insulin through interfering with insulin receptor-mediated signal

transduction34. In agreement with our work results, previous

investigators reported elevated serum levels of such inflammatory

mediators in experimentally-induced diabetes35.

Serious cardiovascular complications of diabetes, including

peripheral arterial disease (PAD) and coronary artery disease (CAD),

are strongly associated with hyperfibrinogenemia36. Fibrinogen

mediates all steps of atherosclerosis, starting from plaque formation

through thromboembolism over a ruptured plaque, leading to

myocardial infarction and other complictions37. A significant elevation

of plasma fibrinogen was evident in the present investigation, which

is in agreement with previous investigations38. In addition,

leucocytosis was previously reported to predict progression of

diabetes39 as well as future CVD40. Our results showing elevated

leucocyte count in diabetic rats come in accordance with previous

investigations41.

Results of the present study showed that combination of LOVA with

either MET or GLIC to rats significantly reduced STZ-induced

pancreatic injury compared to rats receiving either drug alone,

evidenced by suppression of blood glucose level coupled with

improvements of oxidative markers of pancreatic injury including

TBARS, GSH, GST, NO2- and SOD (Table 1). These results

suggest that the beneficial action of LOVA may be related to its

antioxidant power, ameliorating diabetes-induced oxidative and

nitrosative stress. In harmony with our findings, previous

investigators reported protective potential for statins against

oxidative and nitrosative stress42. This is particularly important as

oxidative stress associated with decreased glycemic control has key

effects in the pathogenesis of diabetic complications43. Nitrosative

stress, associated with increased nitric oxide (NO) production, is also

an important event in the pathogenesis of diabetic complications. It

was previously reported that NO production increases in early stages

of diabetes progression, and that inhibition of NO production may be

beneficial in antagonizing STZ-induced diabetes44.

According to our work results, combination of LOVA with MET or

GLIC revealed better improvements of parameters of dyslipidemia,

including serum TGs, TC, LDL and HDL, as compared to LOVA,

MET or GLIC monotherapies (Table 2).

These effects are related to cholesterol-lowering effect of LOVA,

which is an inhibitor of HMG-CoA reductase enzyme that converts

HMG-CoA reductase to mevalonate, the building unit of cholesterol

biosynthesis45. This hypocholesterolemic effect of LOVA is

particularly beneficial in diabetes as it is known to cause

dyslipidemia46. Dyslipidemia associated with diabetes results from

the decreased inhibitory effect of insulin on lipolysis caused by

decreased insulin production or increased insulin resistance.

Stimulated lipolysis in adipocytes causes release of free fatty acids,

the latter being transported to liver, re-esterified to TGs and and

used for assembly of VLDL. Thereafter, serum LDL level increases

and HDL level decreases47. This diabetic dyslipidemia, known as

atherogenic dyslipidemia, is a risk factor for atherosclerosis and

consequently many cardiovascular complications48. This strongly

supports the idea of adding hypocholesterolemic agents with

antidiabetics concerned in the present study.

The present work results showed a significant modulatory potential

of LOVA on MET and GLIC effects on serum CRP and IL-6 levels,

apparent from significant differences between LOVA plus MET or

LOVA plus GLIC groups in comparison with corresponding groups

receiving monotherapies (Table 3). In agreement, previous

investigators reported similar effect of statins on both CRP and IL-6,

mostly attributed to beneficial LOVA effect on dyslipidemia49. Effect

of LOVA on CRP and IL-6 levels is important in diabetes

management as serum CRP and IL-6 levels are indices of acute

phase inflammation and early markers of diabetes incidence50. CRP

is synthesized in the liver under the influence of factors released

from adipocytes in dyslipidemic conditions and can exacerbate

complement-dependent ischemic necrosis leading to myocardial

infarcts51,52. Moreover, the pro-inflammatory cytokine IL-6 is an

important mediator in the pathogenesis of diabetes and diabetic

complications. It increases peripheral insulin resistance34 and

increases atherosclerotic risk in diabetics33,53. Recently, cytokines

were reported not only to be released in response to diabetic

inflammatory state, but also to have a mechanistic role in beta islet

destruction in diabetes progression, and it was also reported that

therapeutic manipulation of cytokines leads to improved glycemic

control in diabetics54. These findings magnify the beneficial role of

LOVA on vascular and even non-vascular diabetic complications.

Results of the present work showed that LOVA combination with

either MET or GLIC significantly improved their suppressant effect on

blood FBG level (Table 3). The antithrombic effect of statins is

attributed to lipid-lowering potential, correcting hemostatic

abnormalities resulting from diabetic dyslipidemia55. The effect of

LOVA on blood FBG level is particularly important in diabetes as

hyperfibrinogenemia is a key factor in atherosclerotic and

inflammatory complications. FBG mediates platelet cohesion and

reversible RBCs aggregation, enhancing atherosclerosis, plaque

formation and formation of thrombus over ruptured plaques.

(6)

Messiha et al. Modulatory Effect of Lovastatin on Therapeutic Efficiency

UK J Pharm & Biosci, 2015: 3(1); 6 dyslipidemia, and stimulate cell proliferation and migration, thus enhancing inflammatory cascades37.

Table 2: Effect of LOVA, MET, GLIC AND their combinations on serum HMG-COA reductase, TGS, TC, LDL-C and HDL-C in STZ-treated

rats

Treatment Regimen

Parameter STZ 7

+ (LOVA plus GLIC STZ6 + (LOVA

plus MET) STZ5 +

GLIC STZ4 +

MET STZ3 +

LOVA STZ2

Control 1

0.55b ± 0.011 0.44b ± 0.010 0.82a,b ± 0.005 0.63b ± 0.012 0.33a,b ± 0.001 1.22a ± 0.016 0.54 ± 0.011 Serum HMG-CoA reductase (ng/ml ) 35.33a,b ± 1.085 31.16a,b ± 0.980 70.55a,b ± 0.493 67.33a,b ± 0.918 40.00a,b ± 1.000 151.16a ± 0.401 94.50 ± 0.562 TGs (mg/dl) 62.16a,b ± 0.477 58.83a,b ± 0.833 90.83a,b ± 0.401 81.16a,b ± 0.654 64.16a,b ± 0.833 112.33a ± 0.843 70.83 ± 0.654 TC (mg/dl) 12.93a,b ± 0.638 8.46a,b ± 0.533 48.16a,b ± 0.792 32.36a,b ± 0.480 15.00a,b ± 1.131 68.10a ± 1.065 20.60 ± 1.281 LDL- C (mg/dl)

42.16a,b ± 0.833 44.83a,b ± 1.013 28.66b ± 0.918 35.33a,b ± 0.614 41.16a,b ± 0.833 14.00a ± 0.577 30.83 ± 0.833 HDL- C (mg/dl)

Data are presented as mean of 6-7 rats ± SEM, 1Control group, received only vehicles, 2STZ control group, administered a single i.p. dose of STZ (50 mg/kg), 3LOVA group, administered LOVA (15 mg/kg/day, p.o.) daily for 30 days after STZ, 4MET group, administered MET (100 mg/kg/day, p.o.) daily for 30 days after STZ, 5GLIC group, administered GLIC (20 mg/kg/day, p.o.) daily for 30 days after STZ, 6LOVA plus MET group, administered LOVA and MET daily for 30 days after STZ, 7LOVA plus GLIC group, administered LOVA and GLIC daily for 30 days after STZ, Statistical analysis was performed by using one way analysis of variance (ANOVA) with turkey’s post hoc comparison test, a

Significantly different from normal control group at p < 0.05, b

Significantly different from STZ control group at p < 0.05.

Table 3: Effect of LOVA, MET, GLIC and their combinations on serum levels of CRP AND IL-6, as well as blood FBG level and WBCS

count in STZ-treated rats

Treatment Regimen

Parameter

STZ 7+ (LOVA

plus GLIC STZ6 + (LOVA

plus MET) STZ5 +

GLIC STZ4 +

MET STZ3 +

LOVA STZ2

Control 1

1.16a,b ± 0.119 0.94a,b ± 0.116 1.86a,b ± 0.009 1.45a,b ± 0.133 2.19a,b ± 0.166 3.36a ± 0.164 0.60 ± 0.004 Serum CRP (ng/ml)

7.36a,b ± 0.080 6.48a,b ± 0.047 8.81a,b ± 0.173 7.40a,b ± 0.134 8.43a,b ± 0.111 10.51a ± 0.166 5.70 ± 0.073 Serum IL-6 (pg/ml )

176.00b ± 0.577 181.75a,b ± 0.629 199.00a,b ± 0.817 220.50a,b ± 0.7637 211.17a,b ± 1.077 567.85a ± 1.922 173.00 ± 0.730 Blood FBG (mg/dl )

7802.50a,b ± 2.500 6998.30a,b ± 1.666 9293.30a,b ± 6.666 8388.30a,b ± 8.333 8315.00a,b ± 15.000 19295.00a ± 5.000 7498.30 ± 1.666 Blood WBCs

(count/mm3)

Data are presented as mean of 6-7 rats ± SEM, 1Control group, received only vehicles, 2STZ control group, administered a single i.p. dose of STZ (50 mg/kg), LOVA group, administered LOVA (15 mg/kg/day, p.o.) daily for 30 days after STZ, 4MET group, administered MET (100 mg/kg/day, p.o.) daily for 30 days after STZ, 5

GLIC group, administered GLIC (20 mg/kg/day, p.o.) daily for 30 days after STZ, 6

LOVA plus MET group, administered LOVA and MET daily for 30 days after STZ, 7LOVA plus GLIC group, administered LOVA and GLIC daily for 30 days after STZ, Statistical analysis was performed by using one way analysis of variance (ANOVA) with turkey’s post hoc comparison test, a

Significantly different from normal control group at p < 0.05, b

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Fig 1: A photomicrograph of pancreas section obtained from normal adult male albino rat, showing normal histological structure of the islands of Langerhans cells (s) as endocrine portion as well as the acini (a) as exocrine portion

Fig 2a: A photomicrograph of pancreas section obtained from adult male albino rats treated with a single dose of streptozotocin (STZ, 50 mg/kg, i.p.), showing atrophy of islands of Langerhans cells (s)

Fig 2b: A photomicrograph of pancreas section obtained from adult male albino rats treated with a single dose of streptozotocin (STZ, 50 mg/kg, i.p.), showing dilatation of blood vessels (V) with perivascular edema and inflammatory cells infiltration (m).

Fig 3a: A photomicrograph of pancreas section obtained from adult male albino rats treated with a single dose of streptozotocin (STZ, 50 mg/kg, i.p.) followed by 30 consecutive days of oral daily administration of lovastatin (LOVA; 15 mg/;g/day), showing atrophy of islands of Langerhans cells (s)

Fig 3b: A photomicrograph of pancreas section obtained from adult male albino rats treated with a single dose of streptozotocin (STZ, 50 mg/kg, i.p.) followed by 30 consecutive days of oral daily administration of lovastatin (LOVA; 15 mg/;g/day), showing multiple number of newly formed pancreatic duct (d).

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Fig 4b: A photomicrograph of pancreas section obtained from adult male albino rats treated with a single dose of streptozotocin (STZ, 50 mg/kg, i.p.) followed by 30 consecutive days of oral daily administration of metformin (MET; 100 mg/kg/day), showing congestion in the interlobular blood vessels (V)

Fig 5: A photomicrograph of pancreas section obtained from adult male albino rats treated with a single dose of streptozotocin (STZ, 50 mg/kg, i.p.) followed by 30 consecutive days of oral daily administration of gliclazide (GLIC; 20 mg/kg/day), showing nearly normal histological structure of islands of Langerhans (S)

Fig 6: A photomicrograph of pancreas section obtained from adult male albino rats treated with a single dose of streptozotocin (STZ, 50 mg/kg, i.p.) followed by 30 consecutive days of oral daily administration of lovastatin (LOVA; 15 mg/kg/day) plus metformin (MET; 100 mg/kg/day), showing normal histological structure of islands of Langerhans (S).

Fig 7: A photomicrograph of pancreas section obtained from adult male albino rats treated with a single dose of streptozotocin (STZ, 50 mg/kg, i.p.) followed by 30 consecutive days of oral daily administration of lovastatin (LOVA; 15 mg/kg/day) plus gliclazide (GLIC; 20 mg/kg/day), showing normal histological structure of islands of Langerhans (S)

Finally, results of the present work revealed that STZ-induced

leucocytosis was improved by LOVA addition to MET or GLIC to

degrees significantly better than either drug alone (Table 3). In

agreement, previous authors reported similar effect of statins on

lecucocyte count and activation56. This effect may be attributed to the

fact that statins have the selective ability to inhibit leukocyte function

antigen-1 by binding to a novel regulatory integrin site57. Additionally,

the anti-inflammatory and cytokine-lowering effect of statins logically

decreases leucocyte activation and proliferation49,58.

Results of histopathological examination were in complete harmony

with biochemical findings, where rats co-treated with LOVA plus MET

or with LOVA plus GLIC after STZ showed almost normalized

pancreatic histology, which is not the case in rats treated with either

drug alone (Fig. 3-7).

5 Conclusion

Results of the present study conclude that combination of LOVA with

conventional antidiabetics has great beneficial effects on

diabetes-induced pancreatic and cardiovascular complications, including

hyperglycemia, oxidative and nitrosative stress, dyslipidemia,

inflammatory disorders, hyperfibrinogenemia and leucocytosis.

These results are promising for further clinical trials and strongly

suggest combining LOVA with antidiabetics in clinical cases of DM.

6 Acknowledgement

Much thanksgiving and appreciation should be directed to Dr. Amal

Khorshid, Assistant Professor of Analytical Chemistry and member of

the Editorial Board, for her extended support and honest advices

helping in publication of the present work.

7 Competing interests

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8 Author’s contributions

Ehab A.M. El-Shoura: Practical work

Basim A.S. Messiha: Manuscript preparation and submission

Amira M. Abo-Youssef: Data collection and resources

Ramadan A.M. Hemeida: Work idea and general supervision

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Figure

Table 1: Effect of LOVA, MET, GLIC and their combinations on serum glucose level and pancreatic contents of TBARS, GSH, NO2-, GST, SOD AND CAT in STZ-treated rats
Table 2: Effect of LOVA, MET, GLIC AND their combinations on serum HMG-COA reductase, TGS, TC, LDL-C and HDL-C in STZ-treated rats
Fig 1: A photomicrograph of pancreas section obtained from normal adult male albino rat, showing normal histological structure of the islands of Langerhans cells (s) as endocrine portion as well as the acini (a) as exocrine portion
Fig 7: A photomicrograph of pancreas section obtained from adult male albino rats treated with a single dose of streptozotocin (STZ, 50 mg/kg, i.p.) followed by 30 consecutive days of oral daily administration of lovastatin (LOVA; 15 mg/kg/day) plus gliclazide (GLIC; 20 mg/kg/day), showing normal histological structure of islands of Langerhans (S)

References

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