Anti-Diabetic and Renoprotective Effects of Menthone in Streptozotocin-Induced Diabetic Nephropathy in Rats

INTRODUCTION

Menthone plays a crucial role in protecting against DN by reducing oxidative stress, inflammation, and hyperglycemia while improving renal function. These findings highlight menthone as a promising candidate for future therapeutic interventions in diabetic nephropathy. With limited treatment options for DN, menthone emerges as a natural compound with therapeutic potential. This study supports its possible clinical application in managing diabetic nephropathy.

Diabetic Nephropathy (DN) emerges as a persistent and incapacitating kidney ailment, evolving as a complication of diabetes mellitus.¹ It assumes a significant role in the escalation of end-stage renal disease globally, impacting individuals with both type 1 and type 2 diabetes. The onset of diabetic nephropathy is intricately linked with prolonged elevation in blood glucose levels, triggering a cascade of interlinked physiological processes within the kidneys.² Streptozotocin (STZ) is a widely used chemical to induce Diabetes Mellitus (DM) in experimental animal models. It primarily targets pancreatic β-cells, leading to hyperglycemia, which in turn contributes to DN over time. The mechanism of STZ-induced DN involves several key pathological pathways like β-cell toxicity, hyperglycemia and glucose toxicity, oxidative stress and mitochondrial dysfunction, inflammatory pathways, renal fibrosis and glomerulosclerosis etc.³ Elevated blood sugar levels lead to glomerular hyperfiltration, renal hypertension, and subsequent thickening of the glomerular basement membrane. Consequently, renal permeability increases, resulting in renal hypoperfusion and proteinuria, notably marked by albumin excretion. With time, chronic inflammation and oxidative stress foster renal fibrosis, compromising kidney function.^4,5 Clinical manifestations include microalbuminuria progressing to proteinuria, hypertension, edema, and a decline in glomerular filtration rate. Diagnosis involves urinalysis, serum creatinine assessment, and monitoring of glomerular filtration rate.⁶ Treatment strategies prioritize stringent glycemic control, blood pressure regulation using medications like ACE inhibitors or ARBs, lifestyle adjustments, and regular monitoring.⁷ The early identification through routine screenings and proactive management of risk factors plays pivotal roles in forestalling or postponing the onset of diabetic nephropathy, emphasizing the significance of holistic care in diabetic individuals. Present therapeutic approaches for diabetic nephropathy focus on managing the progression of renal impairment and alleviating associated complications.⁸ Tight glycemic control serves as a cornerstone, with optimal blood glucose levels pivotal in retarding nephropathy progression.⁹ These medications not only aid in hypertension control but also contribute to reducing proteinuria, thereby preserving renal function. Lifestyle modifications, encompassing a kidney-friendly diet, weight management, and consistent physical activity, complement pharmacological interventions. In instances of advanced diabetic nephropathy, renal replacement therapies like dialysis or kidney transplantation may be contemplated. Regular assessment of kidney function, blood pressure, and glycemic levels is crucial for ongoing diabetic nephropathy management.^10,11 Ongoing research endeavours seek novel therapeutic avenues, emphasizing the necessity of a tailored and comprehensive treatment regimen to address the multifaceted nature of this condition. Early detection, timely intervention, and a holistic care approach remain pivotal in enhancing outcomes and augmenting the quality of life for individuals afflicted with diabetic nephropathy.¹² Menthone, a naturally occurring cyclic monoterpene ketone found in essential oils of various mint species, has gained attention for its potential therapeutic effects in diabetes and diabetic nephropathy.¹³ While previous reports on the direct efficacy of menthone specifically are limited, several studies have investigated the pharmacological properties of mint extracts containing menthone and its derivatives, suggesting promising benefits in managing diabetes and its complications.¹⁴ Menthone has been associated with anti-diabetic properties through its effects on glucose metabolism and insulin sensitivity. One of the studies indicates that mint extracts rich in menthone may enhance insulin secretion from pancreatic beta cells and improve glucose uptake in peripheral tissues, thereby helping to regulate blood glucose levels.^15,16 Moreover, menthone-containing mint extracts have shown potential renoprotective effects in experimental models of diabetic nephropathy. By mitigating oxidative stress, inflammation, and fibrosis in the kidneys, menthone may help attenuate the progression of renal damage and preserve renal function.¹⁷¹⁸) reported that Menthone had potent antibacterial effect on Methicillin Resistant Staphylococcus aureus by affecting cell membrane properties and lipid profile.¹⁸ Most of the studies suggest that mint extracts may reduce proteinuria, inhibit the accumulation of extracellular matrix proteins, and improve renal hemodynamics, thereby delaying the onset and progression of diabetic nephropathy.^19,20 Despite the promising findings from preclinical studies, further research is warranted to elucidate the specific mechanisms of action of menthone and its potential therapeutic utility in clinical settings. This research delves into the multifaceted role of menthone, a naturally occurring compound found in mint extracts, in mitigating diabetes and its renal complications. By exploring different concentrations of menthone, the study seeks to elucidate its dose-dependent effects on various parameters related to diabetes and diabetic nephropathy, including blood glucose levels, insulin sensitivity, biochemical analysis, renal function markers and anti-inflammation activity. Moreover, this study underscores the importance of further investigation into menthone's efficacy, safety, and mechanisms of action, paving the way for its translation into in vivo practice as a promising adjunctive therapy for diabetes and diabetic nephropathy.

MATERIALS AND METHODS

Chemicals

Menthone and streptozotocin were purchased from Sigma Aldrich, USA. TNF-α, IL-1, IL-6, and NF-κB were used in this study by an ELISA kit and procured from Sunred Biological Technology Company, Shanghai. The TRIzol reagent, RNA isolation kit, cDNA synthesis kit, and the SYBR green RT-qPCR kit were sourced from Takara Bio, Japan.

Animal maintenance

A total of 30 male albino Wistar rats (weighing 160-180 g) were used in this proposed study. All the rats were kept in a clean, air-conditioned environment with a temperature of 25ºC, 50% humidity, and a 12:12 hr dark/light cycle. Animals were fed normal diets and given access to drinking water ad libitum. The experimental animal procedure was approved by Institutional Animal Ethics Committee (IAEC).

Animal grouping and treatment

The experiments involving animals were conducted with approval from the appropriate regulatory body. The experimental rats were randomly divided into five groups based on the body weight. Diabetes was induced by intraperitoneal injection of STZ (50 mg/kg BW in 0.1 mmol/l citrate buffer) following 12 hr of fasting. Blood glucose level was monitored three days after STZ administration and then twice a week. The groups were organized as follows:

Group I: Control, Group II: Menthone (50 mg/kg BW) was administered orally for eight weeks,²¹ Group III: Diabetic control STZ injected single dose (50 mg/kg BW in 0.1 mmol/l citrate buffer),¹⁵ Group IV: Diabetic+Menthone (50 mg/kg BW) was administered

orally for eight weeks,

Group V: Diabetic+Gliclazide (10 mg/kg BW) was administered orally for eight weeks.

The body weight of each animal was determined at the end of the study. After eight weeks, the animals fasted for 12 hr, were anesthetized, blood was withdrawn via cardiac puncture and finally sacrificed by decapitation. Blood was centrifuged at 1300 g for separation of serum and plasma separated by respective parameters and stored at 20ºC until assay. Then the urine was collected for urine related parameters.

Kidney function markers test

Serum creatinine (mg/dl), BUN (mg/dl) and urine albumin (mg/ dl) levels were determined following established protocols using autoanalyzer (Hitachi 7600, Japan).²²

Antioxidant activity

The oxidative stress markers such as thiobarbituric acid reactive substances (TBARS),²³ Superoxide Dismutase (SOD),²⁴ Catalase (CAT),²⁵ Glutathione Peroxidase (GPx)²⁶ and reduced glutathione (GSH)²⁷ were evaluated using the standard protocols. The evaluation of lipid peroxidation, specifically the quantification of Thio Barbituric Acid Reactive Substances (TBARS) was performed using known procedures. The sample was diluted with 0.5 mL of double distilled water and thoroughly mixed. Subsequently, 2.0 mL of TBA-TCA-HCL reagent was added. The combination was incubated in a water bath at boiling temperature for 15 min. After cooling, the tubes were subjected to centrifugation at a force of 1000 rpm at 10 min and the liquid above the sediment was measured. The chromophore's absorbance was measured at a wavelength of 535 nm relative to the reagent blank.

0.5 mL of tissue homogenate was diluted to 1.0 mL with water. Added 2.5 mL of ethanol and 1.5 mL of chloroform (chilled), shaken for 90 sec at 4ºC and centrifuged. The assay mixture (1.2 mL sodium pyrophosphate buffer, 0.1 mL of phenazine methosulphate, 0.3 mL of nitroblue tetrazolium) was combined with the enzyme preparation. The reaction was started with 0.2 mL NADH, incubated at 30ºC from 90 sec, and stopped with 1 mL glacial acetic acid. The mixture was shaken with 4 mL n-butanol, allowed to stand for 10 min, centrifuged, and the n-butanol layer's density was measured at 510 nm. 0.1 mL of tissue homogenate was mixed with 0.4 mL of hydrogen peroxide. The reaction was stopped at 15, 30, 45, and 60 s by adding 2.0 mL of dichromate-acetic acid mixture boiling for 10 min, cooled, and the color developed was read at 620 nm.

0.2 mL of tris and 0.2 mL of GSH were mixed, followed by 0.1 mL of H2 O2. The mixture were incubated at 37ºC for 10 min, with a control containing all reagents except homogenate. The reaction was stopped with 0.5 mL 10% TCA, centrifuged, and the supernatant was assayed for GSH using the Ellman method.²⁷ The measurement of reduced Glutathione (GSH) was conducted using the Ellman method. 0.5 mL of tissue homogenate was precipitated with 2.0 mL of 5% TCA. After centrifugation, 2.0 mL of supernatant was mixed, with 1.0 mL of Ellman's reagent and 4.0 mL of 0.3M disodium hydrogen phosphate. The yellow color developed was read at 412 nm.

Anti-Inflammation activity

The inflammatory regulation markers such as TNF-α, IL-1, IL-6, and NF-κB were used in this study by an ELISA kit and procured from Sunred Biological Technology Company, Shanghai, China, following standard procedures.²² In the protocol, 50 µL of the sample and 50 µL of an antibody cocktail were added to the experimental wells and incubated at 37ºC for 1 hr. The wells were then washed with 100 µL of 3,3′,5,5′-Tetramethylbenzidine substrates and incubated for 10 min. Finally, 100 µL of stop solution was added, and the color development was read at OD 450 nm using an ELISA.

Histopathological analysis

Tissue sections of kidney (3-4 μm thick) from all experimental groups were prepared using paraffin embedding. These sections were then stained with Hematoxylin and Eosin (H&E) according to standard procedures to observe cellular alterations.²⁸

Real-time PCR

Total RNA was isolated from the homogenized cardiac material using Ribozol RNA extraction reagent according to the manufacturer's instructions. The reaction mixture had a total volume of 25 µL, which contained 1 µL of total RNA, 1 µL of forward primer and 1 µL of reverse primer, 1 µL of Reverse Transcriptase (M-MLV RT), and 10 µL of single-step SYBR Green PCR Master Mix, plus 20 µL of nuclease-free water. TNF-α, IL-6 and IL-1 genes were compared to Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH), a reference gene. The primer sequences were shown in Table 1. The relative expression level of each gene was determined using the formula 2 (-ΔΔCt) .

Statistical analysis

The study reported all data as mean±standard deviation derived from six rats in each group. Statistical analysis was conducted using One-Way Analysis of Variance (ANOVA) with GraphPad Prism 8 software. Pairwise comparisons between groups were carried out utilizing the least significant difference test, with statistical significance defined as p<0.05.

RESULTS

Menthone effect on glycemic index and renal function of diabetic rats

Four weeks post-STZ injection, diabetic rats demonstrated elevated body weight compared to the control group. However, administration of menthone resulted in decreased body weight and glucose levels. Additionally, diabetic-induced rats exhibited reduced insulin levels, which were increased upon menthone treatment. Furthermore, the HbA1c level was elevated in diabetic animals but was reduced in menthone-treated diabetic rats, as outlined in Figure 1.

Diabetic-induced rats displayed elevated levels of serum creatinine, BUN and urine albumin. However, administration of menthone at a dose of 50 mg/kg BW to diabetic rats significantly decreased (improved in) the levels of serum creatinine, BUN and urine albumin compared to the administration of the conventional medication, gliclazide in Table 2.

Effect of menthone on antioxidant enzyme activity in the kidney of diabetic rats

Renal TBARS levels, reflecting the extent of lipid peroxidation, were observed to be elevated in diabetic-induced animals. However, the menthone-treated group exhibited reduced TBARS levels. Additionally, oxidative enzymes such as SOD and catalase activity were measured, revealing decreased levels in the diabetic group compared to the control. Conversely, the menthone-treated group displayed increased levels of these enzymes compared to the control group, as illustrated in Table 3. Moreover, levels of GPx and GSH were found to be decreased in diabetic rats but increased upon treatment with menthone, surpassing levels observed in the control group. Overall, treatment with menthone in diabetic-induced rats resulted in reduced MDA levels and increased levels of SOD, CAT, GPx, and GSH compared to the control group.

Effect of menthone on inflammatory markers in serum

Levels of TNF-α was found to be elevated in diabetic-induced rats compared to those treated with menthone, where a reduction was observed. Similarly, other inflammatory regulatory markers such as IL-1 and IL-6 showed increased levels in the diabetic group, whereas the menthone-treated groups exhibited reduced levels of these inflammatory regulators. Furthermore, the menthone-treated group demonstrated a significant decline in NF-κB level. In contrast, the diabetic group indicated elevated levels, as depicted in Figure 2. These findings collectively indicate that menthone possesses antioxidant and anti-inflammatory activity in the renal system under diabetic conditions.

Menthone on inflammatory gene expression in kidney tissue

Gene expression analysis revealed significantly upregulated levels of inflammatory targets, including TNF-α, IL-1, and IL-6 in DN-induced rats compared to the control group, as depicted in Figure 3. Conversely, the menthone-treated group exhibited reduced expression levels of TNF-α, IL-1, and IL-6 compared to the DN control group (p<0.05).

Histopathology of diabetic-induced kidney tissues

The study conducted a histopathological analysis of normal kidney tissues, revealing the morphological architecture depicted in Figure 4. Notably, diabetic-induced kidney tissues exhibited ruptured epithelial lining of cells and evident damage primarily in the proximal convoluted tubules. Treatment with menthone resulted in a higher degree of kidney morphology atrophy compared to the diabetic-induced group. Additionally, there was an observable enhancement of normal kidney shapes in the treated group.

DISCUSSION

Diabetic nephropathy, a severe complication arising from diabetes mellitus, remains a predominant cause of End-Stage Renal Disease (ESRD) globally.² It primarily affects individuals diagnosed with either type 1 or type 2 diabetes, characterized by progressive kidney impairment due to sustained hyperglycemia. Prolonged exposure to elevated blood glucose levels leads to damage in the small blood vessels within the kidneys, particularly the glomeruli. This results in increased deposition of extracellular matrix within mesangial cells, leading to mesangial expansion and glomerulosclerosis.^5,6 As the disease advances, the glomerular filtration barrier becomes increasingly permeable, resulting in the leakage of albumin and other proteins into the urine, a condition known as albuminuria or proteinuria. Chronic inflammation, oxidative stress, and activation of profibrotic signaling pathways further contribute to renal fibrosis, ultimately culminating in irreversible kidney damage. The clinical presentation of diabetic nephropathy varies depending on the stage of the disease,⁷ initially manifesting with minimal symptoms that may exacerbate as kidney function deteriorates.

The diagnosis of diabetic nephropathy requires a comprehensive approach involving clinical evaluation, laboratory analyses, and imaging techniques. Measurement of Urinary Albumin-to-Creatinine Ratio (UACR) or Protein-to-Creatinine Ratio (PCR) is employed to assess albuminuria/proteinuria,⁸ while serum creatinine levels and estimated Glomerular Filtration Rate (eGFR) calculations aid in evaluating renal function. Renal ultrasound or other imaging modalities are utilized to assess kidney size, structure, and the presence of renal artery stenosis.^12,13 Over time, extensive research efforts have been dedicated to exploring potential therapeutic interventions for managing diabetic nephropathy, encompassing both pharmacological and non-pharmacological strategies.²⁹ An emerging area of interest in diabetic nephropathy management lies in phytotherapy, which harnesses plant-derived compounds for therapeutic purposes. Phytotherapy has garnered significant attention due to its perceived efficacy, safety, and relatively low cost compared to conventional medications.³⁰ Several plant extracts have been studied for their potential renoprotective effects in diabetic nephropathy. ).

Examples include extracts from plants such as Salvia miltiorrhiza (Danshen), Curcuma longa (Turmeric), Camellia sinensis (Green tea), and Morus alba (Mulberry). These extracts have been shown to possess antioxidant, anti-inflammatory, and antifibrotic properties, which may help mitigate kidney damage associated with diabetes.^31,32 Phytochemicals are bioactive compounds found in plants that exhibit therapeutic potential. Examples include polyphenols, flavonoids, alkaloids, and terpenoids. Studies have investigated the effects of individual phytochemicals, such as resveratrol, quercetin, Epigallocatechin Gallate (EGCG), and berberine, in animal models of diabetic nephropathy.³³ These compounds have been shown to attenuate renal oxidative stress, inflammation, and fibrosis, leading to improvements in kidney function. Menthone, a naturally occurring compound found in various essential oils such as peppermint and pennyroyal, has garnered interest for its potential therapeutic effects in diabetes and its complications, including diabetic nephropathy.³⁴ Menthone exhibits antioxidant properties, which can help combat oxidative stress, a key contributor to the development and progression of diabetic complications, including nephropathy. By scavenging free radicals and reducing oxidative damage, menthone may protect against renal injury in diabetes.

Menthone has been reported to modulate various signaling pathways implicated in the pathogenesis of diabetic nephropathy, including pathways involved in oxidative stress, inflammation, apoptosis, and fibrosis.²⁹ By targeting these pathways, menthone may help prevent or mitigate renal injury in diabetes. Some studies suggest that menthone may have hypoglycemic effects, helping to lower blood glucose levels in diabetes. By improving glucose homeostasis, menthone could potentially reduce the ).U 1 -Enzyme concentration required for 50% inhibition of nitroblue tetrazolium reduction in 1 min; U 2 -μmol of hydrogen peroxide consumed per minute; U3 -μg of glutatione consumed per minute.

risk of diabetic nephropathy and other complications related to hyperglycemia. In this study aimed to understand the role of menthone may exert direct renoprotective effects in diabetic nephropathy. Studies in animal models have demonstrated that menthone treatment can mitigate kidney damage, preserve renal function, and attenuate structural alterations associated with diabetic nephropathy, such as glomerular hypertrophy and tubulointerstitial fibrosis.

Previous studies have reported that the treatment of various drugs resulted in a reduction in glycemic indexes and renal markers.²² Following four weeks post-STZ injection, diabetic rats displayed notable alterations in various parameters compared to the control group. These alterations encompassed elevated body weight and glucose levels, along with reduced insulin levels and heightened HbA1c levels, indicative of impaired glycemic control. Moreover, diabetic-induced rats exhiited elevated levels of markers of renal dysfunction, including serum creatinine, BUN, and urine albumin. However, administration of menthone at a dose of 50 mg/kg body weight to diabetic rats yielded significant improvements in these parameters. Notably, menthone treatment led to decreased body weight and glucose levels, along with increased insulin levels and reduced HbA1c levels, suggesting enhanced glycemic regulation. Additionally, menthone administration resulted in notable reductions in markers of renal dysfunction, surpassing the effects observed with the standard drug, gliclazide.

Furthermore, menthone treatment exerted robust antioxidant effects on the renal system. Elevated renal TBARS levels, indicative of lipid peroxidation, observed in diabetic-induced animals were mitigated by menthone treatment. Chronic hyperglycemia in diabetes triggers immune responses, leading to the activation of inflammatory pathways in the kidneys. This includes the recruitment of immune cells such as macrophages, T cells, and dendritic cells to the renal tissues.³⁵ Moreover, menthone treatment led to increased activity of antioxidant enzymes such as SOD, catalase, GPx, and GSH, surpassing levels observed in the control group. In addition to its antioxidative effects, menthone exhibited potent anti-inflammatory properties. An increased level of TNF-α is a key inflammatory marker that contributes to kidney damage and fibrosis. IL-1 promotes inflammatory responses and worsens renal injury. The pro-inflammatory cytokine IL-6 is associated with diabetic complications and oxidative stress. However, menthone administration significantly reduced these elevated cytokines, demonstrating its anti-inflammatory effects and potential to mitigate renal inflammation in diabetic nephropathy.

Furthermore, menthone adsministration resulted in a significant decline in NF-κB level, indicating its ability to modulate the inflammatory response in diabetic nephropathy. Histopathological examination of kidney tissues from DN rats reveals a spectrum of structural abnormalities, including glomerular hypertrophy, tubulointerstitial fibrosis, vascular changes, inflammatory infiltrates, and other pathological features.^36,37 Histopathological examination further corroborated the beneficial effects of menthone treatment on renal morphology. Diabetic-induced kidney tissues exhibited significant structural abnormalities, including ruptured epithelial lining of cells and proximal convoluted tubular damage. Conversely, menthone-treated groups demonstrated improved kidney morphology, characterized by reduced atrophy and enhanced preservation of normal tissue architecture.

Previous study stated that the effect of ursolic acid regulates oxidative stress and involved in inflammatory responses which are strongly corelated with diabetic conditions.³⁸ Western blot analysis revealed significantly upregulated levels of inflammatory targets, including TNF-α, IL-1, and IL-6, in DN rats compared to the control group. Conversely, the menthone-treated group exhibited reduced expression levels of these inflammatory markers compared to the standard drug group. Overall, these findings collectively underscore the therapeutic potential of menthone in the management of diabetic nephropathy. Its multifaceted effects, encompassing antioxidative, anti-inflammatory, and renoprotective properties, position menthone as a promising therapeutic agent for battling the renal complications associated with diabetes.

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CONCLUSION

This study demonstrates that menthone exhibits renoprotective effects in STZ-induced DN rats. The diabetic rats exhibited characteristic signs of DN, including elevated glucose and glycosylated hemoglobin levels, reduced insulin levels, increased oxidative stress markers, decreased antioxidant levels, elevated levels of blood creatinine, BUN, urine albumin, and heightened pro-inflammatory cytokines in renal tissues. However, oral administration of menthone at doses of 50 mg/kg for eight weeks resulted in significant reductions in hyperglycemia, oxidative stress, and inflammation, along with enhancements in antioxidant levels in the kidneys. These findings suggest that menthone possesses potent renoprotective properties by improving renal function, reducing oxidative stress, and mitigating inflammation in diabetic rats. Consequently, menthone emerges as a promising therapeutic agent for the treatment of diabetic nephropathy. Further investigation through clinical trials is warranted to validate the efficacy and safety of menthone as a potential treatment option for diabetic nephropathy. Research should assess the long-term safety and efficacy of menthone treatment in chronic diabetic conditions. Comparing menthone's efficacy with standard anti-diabetic and renoprotective drugs to evaluate its potential as a complementary or alternative therapy.

SUMMARY

This study highlights the renoprotective effects of menthone in STZ-induced DN in rats. Diabetic rats displayed typical DN symptoms, including increased glucose, glycosylated hemoglobin, creatinine, blood urea nitrogen, urine albumin, oxidative stress markers, and pro-inflammatory cytokines, along with reduced insulin and antioxidant levels. Oral administration of menthone for eight weeks significantly improved renal function by reducing hyperglycemia, while increasing kidney antioxidant capacity, reduces oxidative stress and protecting kidney cells. These findings suggest menthone's potential as a therapeutic agent for diabetic nephropathy, warranting further validation through clinical trials.

Funding

The author received no financial support for the research, authorship and/or publication of this article.

Conflict of Interest

The authors declare that there is no conflict of interest.

Data Availability

The data can be obtained from the corresponding author upon reasonable request.

Ethics Statement

The experimental methodology was permitted by the Institute Animal Ethics Committee of Luohe Medical College，Luohe City, Henan Province, China.

Streptozotocin; Superoxide Dismutase; GPx: Glutathione Peroxidase; GSH: Reduced Glutathione; TNF-α: Tumor Necrosis Factor Alpha;

Kappa-Light-Chain-Enhancer of Activated B Cells; TBARS: Thiobarbituric Acid Reactive Substances; BUN: Blood Urea Nitrogen.