Minocycline with Aspirin: An Approach to Attenuate Diabetic Nephropathy in Rats

Abstract

Degradation of extracellular matrix (ECM) by enhanced production of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) in diabetes leads to nephropathy. Cyclooxygenases (COX) further increase levels of these MMPs. The objective of present study was to inhibit MMP-2 and MMP-9 by combination of minocycline and aspirin to treat diabetic nephropathy. Diabetes was induced in male Wistar rats by streptozotocin (STZ, 55 mg/kg i.p.). Four weeks after diabetes induction, the rats were treated with minocycline (50 mg/kg, p.o.), aspirin (50 mg/kg, p.o.), or minocycline (50 mg/kg, p.o.) plus aspirin (50 mg/kg, p.o.) for a period of 4 weeks. At the end of eighth week fluid input, urine output, and renal function tests were carried out for diagnosis of diabetic nephropathy. Renal hypertrophy was measured and histopathology was done to evaluate renal damage. Diabetes produced significant loss of body weight, polyuria, polydipsia, hyperglycemia, and increase in blood pressure. Serum creatinine, urea, and blood urea nitrogen levels were found to be increased significantly in the STZ group diabetic rats. Treatment with combination of minocycline and aspirin significantly prevented the rise in creatinine, urea, and blood urea nitrogen levels and increased creatinine clearance. Image analysis of kidneys revealed that collagen level was significantly decreased in combined treated group when compared with control. Results of present study suggest that MMP-2 and MMP-9 inhibition in presence of COX inhibitor prevents the development of experimental diabetic nephropathy in rats and can be a potential approach for the treatment.

• extracellular matrix
• diabetic nephropathy
• matrix metalloproteinase-2
• matrix metalloproteinase-9

INTRODUCTION

More than 30% of diabetes mellitus (DM) patients develop clinically evident diabetic nephropathy 10–20 years after the onset of diabetes¹ and diabetic nephropathy is now the most common cause of end-stage renal disease requiring renal transplantation or chronic hemodialysis.² Despite the benefits derived from the current therapeutics for diabetic nephropathy, mainly strict control of glucose, blood pressure, and blockade of the renin–angiotensin system, these strategies still provide imperfect protection against renal progression.^3,4 This imperfection points to the need for newer therapeutic strategies that have potential to affect primary mechanisms contributing to the pathogenesis of diabetic nephropathy.⁵ These include increased oxidative stress, renal polyol formation, advanced glycated end-products accumulation, and presclerotic cytokines such as transforming growth factor beta-1 (TGF-β1). These pathways ultimately lead to increased renal albumin permeability and extracellular matrix (ECM) accumulation, which result in increasing proteinuria, glomerulosclerosis, and tubulointerstitial fibrosis.

The accumulation of ECM within the kidney is an ultrastructural hallmark of diabetic nephropathy and is directly linked to a decline in renal function.⁶ This increase of ECM can result from either increased synthesis and decreased degradative activity or both. Several studies have shown that high glucose concentration increases synthesis of ECM proteins in both mesangial and tubular epithelial cells.⁷ Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidase that mediate the degradation or remodeling of the ECM.⁸ The ECM is a multifunctional complex of proteins and proteoglycans assembled in a highly organized manner that contributes to the structural integrity of cells and tissue within an organ system.⁹ The basement membrane which provides structural support to the vasculature is composed of ECM molecules such as type IV collagen, laminin, and fibronectin.¹⁰ Various factors are involved in maintaining the integrity of the ECM and the tissues it supports. However, in certain pathological circumstances the ECM is modulated such that the structure of the tissues becomes damaged or destroyed.^11,12 Two members of the MMP family, matrix metalloproteinase-2 (MMP-2, 72 kD gelatinase/gelatinase A) and matrix metalloproteinase-9 (MMP-9, 92 kD gelatinase/gelatinase B), degrade the ECM components of the basement membrane. Their substrates include types IV and V collagen, fibronectin, elastin, and denatured interstitial collagens.¹³ Two groups of extracellular proteinases that have been shown to play a role in the renal revascularization seen in diabetic nephropathy are the MMP-2 and MMP-9. Plasmin in turn degrades components of the matrix and can be involved in the activation of the latent MMPs.

We hypothesized that remodeling of ECM could be attenuated by minocycline-induced inhibition of MMP-2 and MMP-9 and further it can be potentiated by aspirin because of its Cyclooxygenases (COX) and tissue plasminogen activator (tPA) inhibitory action. The basis for this hypothesis was a case report by Ahuja¹⁴ on an incidental reduction of glomerulonephritis proteinuria by 70% with doxycycline treatment. Earlier, we have reported attenuation of diabetic neuropathy¹⁵ and diabetic retinopathy¹⁶ by this combination in rats. In the present study, we have targeted MMP-2 and MMP-9 over-activation in diabetic nephropathy using a known MMP-2 and MMP-9 inhibitor, minocycline with a nonselective COX inhibitor aspirin. Although aspirin is nonselective COX inhibitor, it has been selected because of its inhibitory effect on tPA. This finding may indicate the potential benefit of minocycline along with aspirin treatment in easing the chronic consequences of diabetic nephropathy.

MATERIALS AND METHODS

Chemicals and Drug Solution Preparation

Streptozotocin (STZ) was purchased from Sigma (St. Louis, MO, USA). Minocycline was procured from US Vitamins, Mumbai, India, as a gift sample. Aspirin was purchased from Central Drug House, Mumbai, India. Glucose oxidase–peroxidase (GOD/POD) glucose kit was purchased from Erba Diagnostics, Mumbai, India. All other chemicals were purchased from Merck (Mumbai, India). Minocycline was dissolved in distilled water. Aspirin was suspended in aqueous solution of 0.5% carboxymethyl cellulose. STZ was freshly dissolved in ice-cold citrate buffer (pH 4.5) solution.

Animals

Male Wistar rats (210–250 g) were purchased from the Haffkine Institute (Mumbai, India) and were housed at a temperature of 25 ± 1°C and relative humidity of 45–55% in a clean environment under 12:12 h light and dark cycle. The animals had free access to food pellets and filtered water was made available ad libitum. The research protocol was approved by Institutional Ethical Committee (IEC) of School of Pharmacy and Technology Management, NMIMS University, Mumbai, constituted under Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA).

Induction of Diabetes

A single dose (55 mg/kg, i.p.) of STZ was used for induction of diabetes in rats. Age-matched control rats received the equal volume of vehicle. Diabetes was confirmed after 48 h of STZ injection and again on weekly basis during the experiment. Plasma glucose levels were estimated using GOD/POD kit and rats with plasma glucose level >350 mg/dL were considered for further studies.

Experimental Design and Drug Treatment

Four weeks after the diabetic induction, treatments were given for further 4 weeks (fifth, sixth, seventh, and eighth weeks). After 4 weeks, normoglycemic and diabetic rats were randomly divided into experimental groups and treated with drugs as follows:

• Diabetic vehicle treated Group 1 (DB + DW) was treated with distilled water 4 mL/kg, p.o.).

• Diabetic vehicle treated Group 2 (DB + 0.5% CMC) was treated with 0.5% carboxymethyl cellulose solution (1 mL/kg, p.o.).

• Diabetic Group 3 (DB + MINO) was treated with minocycline alone (MINO, 50 mg/kg, p.o.).

• Diabetic Group 4 (DB + ASP) was treated with aspirin alone (ASP, 50 mg/kg, p.o.).

• Diabetic Group 5 (DB + MINO + ASP) was treated with combination of minocycline (MINO, 50 mg/kg, p.o.) and aspirin (ASP, 50 mg/kg, p.o.).

• Age-matched Normal Group 6 was untreated.

Renal Function Tests

At the end of the eighth week, rats were kept individually in metabolic cages for 24 h to collect urine for the measurement of urine output and renal function. Renal function was assessed by measuring plasma and urine levels of creatinine, urea, and urine albumin excretion using semi-auto-analyzer (Erba Chem-5 plus; Transasia, Mumbai, India). Creatinine clearance was measured as an index of glomerular filtration rate (GFR).

Systemic Blood Pressure

Eight weeks after STZ or vehicle, rats were anesthetized with pentobarbital sodium and body temperature was maintained at 37 ± 1°C. Systemic blood pressure was monitored via a catheter inserted into the femoral artery. Blood pressure was recorded digitally through a data acquisition system (Power Lab system, AD Instruments, New South Wales, Australia). Mean arterial pressure (MAP) was calculated using the following formula: MAP = diastolic pressure + 1/3 (systolic pressure – diastolic pressure).

Renal Hypertrophy and Histopathology

After completion of MAP recording, rats were perfused via the abdominal aorta with 0.1 mol/L PBS (150 mL [pH 7.4], 180–220 mmHg) for 1–2 min to remove circulating blood. The right kidney was then removed, weighed, and kidney/body weight ratio calculated. Data were expressed as relative organ weight of one kidney to 100 g of total body weight. Paraformaldehyde-fixed (4%) tissue was embedded in paraffin and sectioned at 5 μm for morphologic analysis. The kidney specimens were fixed in 4% paraformaldehyde solution and embedded in paraffin. Sections were cut at 4 mm with a microtome and deparaffined with xylene. They were stained with Masson's trichrome stain and hematoxylin and eosin (H&E) using standard histological procedure. Stained kidney sections were observed under a light microscope.

Index of Tubulointerstitial Fibrosis

Masson's trichrome-stained sections were examined and the degree of tubulointerstitial fibrosis determined using a semiquantitative scoring method: 0 = no lesions showing cell infiltration and fibrosis, 1 = minimal injury (single focus of lesion), 2 = mild injury (more than two isolated foci), 3 = moderate injury (more than five isolated foci), and 4 = severe injury (more than 10 isolated foci or diffuse infiltration and fibrosis).

Histology of Renal Artery

Paraffin-embedded cross-sections (5 μm thick) of renal artery were stained with hematoxylin and eosin (H&E) solution (Sigma), and Elastin Van-Giesen staining was performed using the Elastic Stain kit to stain collagen fibers. Six sections of artery from all animals were used for collagen fiber analysis. Images were captured using a CCD camera mounted in a microscope (Motic^®). Area of red-colored collagen was measured using Scion Image software (NIH, Bethesda, Maryland, USA). Area of collagen was represented as percent value of total area measured.

SDS-PAGE Zymography

Rats from each group were anesthetized deeply and then perfused transcardially with ice-cold PBS, pH 7.4. The left kidneys were removed quickly and frozen immediately on dry ice, and stored at –80°C. Tissue samples were homogenized, centrifuged, and supernatant was collected. Prepared protein samples were loaded and separated by 10% Tris-glycine gel. After separation by electrophoresis, the gel was stained with 0.5% Coomassie Blue R-250 for 30 min and then destained appropriately. MMP-2 (72 kD) and MMP-9 (92 kD) bands were confirmed by comparison with commercially available molecular weight marker.

Statistical Analysis

Data are expressed as mean ± SD. Statistical analysis was performed using Graph Pad Prism (version 4.0, Graph Pad Inc., San Diego, CA, USA) software. For multiple comparisons, one-way analysis of variance (ANOVA) was used. In case ANOVA shows significant differences, post hoc analysis was performed with Dunnet post hoc test; p < 0.05 was considered statistically significant.

RESULTS

Plasma Glucose Levels and Body Weight

STZ-induced diabetic rats showed approximately fivefold increase in the blood glucose levels after STZ administration. Out of 40 animals, 4 animals were discarded from the study due to less increase in BSL (i.e., less than 350 mg/dL). Rest of the 36 animals showed elevated BSL which was consistent throughout the study period (Table 1). Diabetic rats showed significant decrease in body weight as compared to age-matched normoglycemic rats (Table 1). Treatment with MINO, ASP or combination of MINO and ASP did not produce any change in plasma glucose levels (Table 1).

Table 1. Effects of 4-week treatment with MINO, ASP, and MINO in combination with ASP on renal function tests

Groups	NORMO	DB + DW	DB + ASP	DB + MINO	DB + MINO + ASP	DB + 0.5% CMC
Plasma glucose (mg/dL)	113 ± 7.16	421 ± 14.65	412 ± 20.77	421 ± 20.80	409 ± 16.83	424 ± 28.21
Body weight (g)	262.4 ± 14.10	217.4 ± 7.96	211.2 ± 11.03	215 ± 9.41	214.8 ± 13.24	210.2 ± 12.26
Food intake (g)	32.2 ± 5.2	65.6 ± 6.7	63.2 ± 7.5	64.6 ± 4.5	62 ± 5.1	62.6 ± 5.5
Water intake (mL)	35.4 ± 4.9	123.6 ± 7.3	108.2 ± 8.1	99.4 ± 9.1*	81.8 ± 6.1**	121.4 ± 13
Urine output (mL)	9.44 ± 1.5	42.44 ± 5.1	37.44 ± 3.2	32.28 ± 4.3*	27 ± 3.6**	39.36 ± 4.8
Urine albumin (mg/dL)	4.02 ± 0.35	12.96 ± 1.03	11.06 ± 1.10	9.54 ± 1.03*	7.28 ± 0.63**	13.06 ± 1.04
Serum creatinine (mg/dL)	0.326 ± 0.03	1.406 ± 0.07	1.2 ± 0.12	1.016 ± 0.11*	0.776 ± 0.07**	1.352 ± 0.08
Blood urea nitrogen (mg/dL)	24.56 ± 1.48	61.16 ± 3.11	53.52 ± 3.03	49.18 ± 3.02*	43.84 ± 3.74**	63.46 ± 3.43

Note: The values are given as mean ± SD. *p < 0.01 and **p < 0.01 when compared with vehicle treated diabetic group.

Renal Function Tests

Diabetic rats exhibited marked polyuria, increased urinary albumin excretion, high serum creatinine, as well as blood urea nitrogen. Chronic treatment with MINO + ASP significantly reduced diabetic proteinuria and polyuria, and increased serum creatinine and blood urea nitrogen (Table 1). Creatinine clearance was also significantly improved following the administration of MINO + ASP to diabetic rats compared with untreated diabetic rats (Figure 1). Marked renal hypertrophy was observed in diabetic group. Kidney to body weight/100 g ratio was significantly higher in diabetic group (0.54 ± 0.05, p < 0.05) compared to normoglycemic group (0.32 ± 0.02). Treatment with MINO + ASP attenuated renal hypertrophy significantly when compared with diabetic group (0.41 ± 0.03, p < 0.05, Figure 2). Mean arterial pressure was decreased in diabetic group (82 ± 6.4 mmHg) compared to normoglycemic (93 ± 4.4 mmHg) group and attenuated significantly by the MINO + ASP treatment (90 ± 3.2, p < 0.05, Figure 3). Treatment with MINO per se and ASP per se reduced severity of nephropathy but was not significant.

Figure 1. Effects of 4-week treatment with MINO, ASP, and MINO in combination with ASP on creatinine clearance. The values are given as mean ± SD. *p < 0.05, **p < 0.01 when compared with vehicle treated control group.

Figure 2. Effects of 4-week treatment with MINO, ASP, and MINO in combination with ASP on renal hypertrophy. The values are given as mean ± SD. **p < 0.01 when compared with vehicle treated control group.

Figure 3. Effect of 4-week treatment with MINO, ASP, and MINO in combination with ASP on mean arterial pressure. The values are given as mean ± SD. *p < 0.05 when compared with vehicle treated diabetic group.

Renal Histology

Histological slides of kidneys from the diabetic group showed markedly severe destruction in glomerular and tubulointerstitial lesions such as glomerular sclerosis, atrophy, interstitial expansion, and interstitial cellular infiltration as compared with those of the control group. Also, collagen deposition was prominent and tubular spaces were obstructed. In the diabetic rats with the MINO + ASP, general morphology of glomerulus and tubulointerstitial lesions was much improved and showed quite normal appearance.

Tubulointerstitial Fibrosis Index

The kidneys of normoglycemic rats exhibited normal cortical and medullary morphology. Mild tubulointerstitial fibrosis, characterized by accumulation of ECM, tubular dilatation, and atrophy, was observed in the vehicle treated diabetic animals. The tubulointerstitial fibrosis index was 1.6 ± 0.24 in renal cortex and medulla of vehicle treated diabetic rats (Table 2). Treatment with MINO plus ASP significantly reduced tubulointerstitial fibrosis (0.6 ± 0.25, p < 0.10) compared to vehicle treated diabetic control.

Table 2. Effects of 4-week treatment with MINO, ASP, and MINO in combination with ASP on renal artery collagen level and renal tubulointerstitial fibrosis index

Group	% Collagen	Tubulointerstitial fibrosis index
NORMO	26.9 ± 1.19	0
DB + DW	37.4 ± 2.73	1.6 ± 0.24
DB + MINO	35.2 ± 3.15	1.4 ± 0.24
DB + ASP	33.9 ± 2.87	1.0 ± 0.31
DB + ASP + MINO	30.8 ± 3.21*	0.6 ± 0.04*
DB + 0.5% CMC	38.1 ± 2.74	0

Note: The values are given as mean ± SD. *p < 0.01 when compared with vehicle treated diabetic group.

Histology of Renal Artery

Diabetes causes increase in collagen level in arteries. Diabetes increased collagen level significantly (37.4% ± 2.73) when compared with normoglycemic group (26.9% ± 1.19, p < 0.05). Treatment with MINO per se and ASP per se reduced collagen level slightly but not significantly. Four-week treatment with MINO in combination with ASP significantly decreased (30.8 ± 3.21, p > 0.01) collagen level when compared with diabetic group (37.4 ± 2.73, Table 2).

SDS-PAGE Zymography

Zymogram of kidney homogenate showed high level of 72 kD protein and 92 kD protein in vehicle treated diabetic animals compared to normal animals (Figures 4 and 5). Treatment with MINO per se, ASP per se, and MINO plus ASP reduced the increased level of 72 kD and 92 kD protein. Reduction in 72 kD and 92 kD protein level is more with MINO plus ASP treated group compared to MINO per se and ASP per se treated group (Figures 4 and 5).

Figure 4. Effect of 4-week treatment with MINO, ASP, and MINO in combination with ASP on renal protein (72 kD) level. (a) Loading control; (b) DB + ASP + MINO; (c) DB + MINO; (d) DB + ASP; (e) DB + 0.5 % CMC; (f) DB + DW; (g) NORMO.

Figure 5. Effect of 4-week treatment with MINO, ASP, and MINO in combination with ASP on renal protein (92 kD) level. (a) Loading control; (b) DB + DW; (c) DB + MINO; (d) DB + ASP + MINO; (e) DB + ASP; (f) NORMO; (g) DB + 0.5% CMC.

DISCUSSION

Diabetic nephropathy is the most common single cause of renal insufficiency. Glomerular changes, such as capillary basement membrane thickening, mesangial proliferation, and nodular glomerulosclerosis, are pathognomic for diabetic nephropathy.¹⁷ Tubulointerstitial fibrosis is a predictor of progressive renal failure.¹⁸ MMPs have been proposed as important factors driving fibrosis in the glomerulus in various renal diseases, including diabetic nephropathy.¹⁹ MMP-2 and MMP-9 belong to the type IV collagenase family of MMPs and are responsible for the degradation of collagen type IV, V, VII, elastin, and fibronectin. Collagen-IV has been identified as the major collagenous component of glomerular basement membrane and ECM.²⁰ As the pathophysiologic actions of MMPs are quite complex and the mechanisms of remodeling appear to be heterogeneous, an abnormal degradation and deposition of ECM may play an important role in the development of structural alterations in glomeruli that contribute to proteinuria.

It is conceivable that abnormalities in collagen-IV degradative pathways take part in the progression of diabetic nephropathy. A role for MMPs in the alteration of basement membrane permeability has been postulated in patients with membranous GN, which reveals elevated levels of TGF-β and collagenase activity in urine.²¹ Moreover, collagen-IV and fibronectin, which are the major components of ECM, and MMP and TIMP expressions were all altered in diabetic and other nephropathies.^22–24 Expansion of ECM in tubulointerstitium and glomerulus is the typical pathological feature of diabetes.²⁵ MMPs also seem to play a crucial role in epithelium–mesenchyme interactions during renal development and determination of nephron number, which can lead to development of chronic renal disease later in life.²⁶

McMillan et al.²⁷ have shown that increased glomerular epithelial cell MMP-9 expression correlates with the period of proteinuria associated with Heymann nephritis and that a direct link exists between glomerular epithelial cell proteolytic activity and loss of glomerular permselectivity. Similarly, MMPs can increase microvascular endothelial permeability. It is also shown that elevated plasma MMP-9 concentration precedes and may predict the development of microalbuminuria in type II DM.²⁸ Furthermore, treatment of nephrotic animals with a synthetic MMP inhibitor has resulted in a reduction of mesangial cell proliferation, ECM deposition, glomerular hypertrophy, and proteinuria.²⁹ These studies strongly suggest a role played by the MMPs in renal diseases such as diabetic nephropathy.

We hypothesized that inhibition of MMP-2 and MMP-9 can attenuate diabetic nephropathy. In our study, 4-week treatment with ASP per se, MINO per se, and ASP plus MINO did not alter blood sugar level and body weight compared to vehicle treated diabetic group which showed that treatment with combination is attenuating diabetic nephropathy through other than glucose-lowering mechanism. This may be due to altered expression of the MMPs and degradation of ECM proteins in the presence of minocycline which is a potent nonselective inhibitor of MMPs and aspirin. STZ-injected rats demonstrated typical characteristics of DM such as hyperglycemia, polyuria, growth retardation, and an increase in urinary albumin excretion. Eight weeks of diabetes induced significant defect in renal function tests, renal hypertrophy, and renal collagen level. Increased blood urea nitrogen and serum creatinine in diabetic rats indicates progressive renal damage which is taken as an index of altered GFR in diabetic nephropathy.³⁰ Treatment with MINO in combination with ASP attenuated renal function tests and renal hypertrophy.

Previous studies have shown that MMPs are involved in the remodeling of the ECM and increase collagen deposition.³¹ This leads to renal damage and decline in renal function. In the present study, treatment with combination of minocycline and aspirin reduces collagen deposition. This reduction in collagen deposition was more than per se treated group. This reduced remodeling of ECM leads to normalization of vascular structure and reversal of vascular reactivity. Impaired endothelium-dependent vasodilatation has been demonstrated in various vascular beds in diabetic patients and diabetic animals.³² Improved vascular bed leads to amelioration of renal damage which is supported by the histological findings suggesting marked attenuation in renal structures in MINO plus ASP treated group.

In our study, we used MINO in combination with ASP to reduce increased concentration of MMP-2 and MMP-9 in diabetic rats. MINO is a direct inhibitor of MMP-2 and MMP-9. ASP can inhibit MMP-2 and MMP-9 biosynthesis by inhibiting COX-2 and by inhibiting tPA. In diabetics, hyperglycemia induces fibronectin (Fn) overexpression which degrades into proangiogenic Fn fragments (Fn-f). Fn-f has been shown to stimulate tPA-catalyzed plasminogen activation; the plasmin produced then activates proform of MMP-2 and MMP-9.³³ Results of zymography suggest that level of 72 kD protein and 92 kD protein which are molecular weights of MMP-2 and MMP-9 are reduced with treatment of MINO plus ASP. Further, these reductions were higher as compared to per se treated groups. Treatment with MINO in combination with ASP attenuated renal function tests which suggested that enhanced inhibition of MMP-2 and MMP-9 by the combination is responsible for improvement.

Tetracycline-like minocycline and doxycycline have been shown to inhibit MMP activity; doxycycline suppresses the activation of proMMP-2 and inhibits the mononuclear phagocyte expression of MMP-9 mRNA. However, Kaito et al.³⁴ reported that doxycycline does not affect MMP-2 production. Although human trials with tetracycline were reported controversially, minocycline has been shown to inhibit MMP-2 and MMP-9 activity in most cases.¹³

One of the most important aspects of the MMP inhibitors has been the poor correlation between very promising preclinical studies with these drugs and the complete failure of the same drugs when tested clinically. One possible reason could be the vast difference in tumor biology between mouse models and human cancer. As the majority of patients in all of the MMP inhibitor phase III trials corresponded to unresponsive later stage, it is perhaps not surprising that clinical testing of MMP inhibitors failed to replicate the positive results from preclinical studies. Although MMP inhibitors showed poor clinical results, the results of the study by Ahuja¹⁴ on an incidental reduction of glomerulonephritis proteinuria by 70% with doxycycline treatment in patients revealed therapeutic benefit.

In conclusion, this study demonstrated in experimental diabetic nephropathy that the renoprotective effects of treatment with combination of MINO and ASP that attenuates the accumulation of collagen may, in part, occur via inhibition of MMP-2 and MMP-9. Our study suggests beneficial effects of treatment with combination of MINO and ASP in experimental diabetic neuropathy in rats and can be a potential approach for the treatment.

Declaration of interest:The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.