Renoprotective and antioxidative effects of methanolic Paederia foetida leaf extract on experimental diabetic nephropathy in rats

Author’s Accepted Manuscript Renoprotective and Antioxidative Effects of Methanolic Paederia foetida Leaf Extract on Experimental Diabetic Nephropathy in Rats M.P. Borgohain, L. Chowdhury, S. Ahmed, N. Bolshette, K. Devasani, T.J. Das, A. Mohapatra, M. Lahkar www.elsevier.com/locate/jep

PII: DOI: Reference:

S0378-8741(17)30249-0 http://dx.doi.org/10.1016/j.jep.2017.01.035 JEP10682

To appear in: Journal of Ethnopharmacology Received date: 9 May 2016 Revised date: 8 December 2016 Accepted date: 18 January 2017 Cite this article as: M.P. Borgohain, L. Chowdhury, S. Ahmed, N. Bolshette, K. Devasani, T.J. Das, A. Mohapatra and M. Lahkar, Renoprotective and Antioxidative Effects of Methanolic Paederia foetida Leaf Extract on Experimental Diabetic Nephropathy in Rats, Journal of Ethnopharmacology, http://dx.doi.org/10.1016/j.jep.2017.01.035 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Renoprotective and Antioxidative Effects of Methanolic Paederia foetida Leaf Extract on Experimental Diabetic Nephropathy in Rats Borgohain M. P.1, Chowdhury L.1, Ahmed S.1, Bolshette N.3, Devasani K.4,Das T. J.1, Mohapatra A.1, Lahkar M.2,3* 1

Department of Pharmacology & Toxicology, National Institute of Pharmaceutical Education

and Research, Guwahati, Assam, India. 2

Depertment of Pharmacology, Gauhati Medical College & Hospital, Guwahati, Assam,

India. 3

Institute Level Biotech-hub, National Institute of Pharmaceutical Education & Research,

Guwahati, Assam, India. 4

Department of Pharmacology and Toxicology, Bombay College of Pharmacy, Mumbai,

Maharashtra, India. *Corresponding Author: Prof. Mangala Lahkar, HOD Department of Pharmacology, Gauhati Medical College & Hospital, Bhangagarh, Guwahati-32, Assam, India. Contact No. +918811848404, E-mail: [email protected]

Abstract: Ethnopharmacological relevance Paederia foetida Linn. (Family: Rubiaceae) is widely used as natural remedy for diabetes mellitus by the Nepali and Lepcha tribes of Sikkim and Darjeeling Himalayan region. The plant is administered to a diabetic individual in the form of leaf infusion for 2-3 weeks. Therefore, we investigated the effects of methanolic leaf extract of Paederia foetida (MEPF) on alloxan (ALX) induced diabetic renal oxidative stress and NF-kB dependent renoinflammatory events in rat. Method Effects of MEPF on blood glucose, glomerular filtration rate (GFR), serum and oxidative stress markers were evaluated in ALX induced diabetic wistar rats. Enzyme linked immunosorbent assay (ELISA) was carried out to estimate serum IL-6, IL-1β, TNF-α and

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renal tissue NF-kB p65 levels. MEPF treatment was given to the diabetic rats at a dose of 250 and 500 mg/kg body weight (b.w.). Results & Discussion MEPF treatment significantly reduced hyperglycaemia, serum creatinine, blood urea nitrogen (BUN), bilirubin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), triglycerides (TRIGs), and total cholesterol (TCHOL) levels in the diabetic rats, whereas it significantly restored GFR and serum albumin level. The activity of enzymatic and nonenzymatic antioxidant machineries was remarkably augmented by MEPF treatment. Likewise, it also significantly lowered lipid peroxidation which was evidenced by decreased concentration of thiobarbituric reactive substances (malondialdehyde, MDA) in the renal tissue of treated diabetic groups. Moreover, MEPF treated animals exhibited low serum concentrations of IL-6, IL-1β and TNF-α compared to diabetic control rats. It showed a dose dependent inhibition of NF-kB activation in the diabetic kidney. But the effects were more prominent at a dose of 500 mg/kg. Histopathological examinations also confirmed its nephroprotective action during diabetes. Conclusion MEPF treatment mitigates oxidative stress and suppress renal inflammation via inhibition of NF-kB in diabetic kidney in early progressive diabetic nephropathy.

Keywords: Diabetic nephropathy; Oxidative stress; Inflammation; Alloxan; Paederia foetida; NF-kB

1. Introduction Diabetes mellitus (DM) is an increasingly global health problem today. In 2013, 382 million people suffered from DM around the globe and type 2 accounts for 90% of the cases with no gender disparity (Shi and Hu, 2014; Vos et al., 2013). The global diabetic population is projected to increase to 439 million by 2030; which is equal to 7.7% of total adult population (Chen et al., 2012). DM is characterized by altered glucose, lipid and protein metabolism. The altered metabolism results in microvascular complications; notably including nephropathy, neuropathy and retinopathy (Giacco and Brownlee, 2010; Kitada et al., 2010). One third of type 2 diabetics suffer from nephropathy and have a high cardiovascular mortality and morbidity (Thomas and Karalliedde, 2015). Diabetic nephropathy (DN) is the single most important cause of end stage renal disease (ESRD) worldwide and contributes to 2

40% of the new cases in both Asian and Western countries (Keane et al., 2003). Studies on DN, revealed that oxidative stress on kidney and chronic inflammation; both equally contribute to its development and progression (Brownlee, 2005). NF-kB linked inflammatory cascade is reported to have key role in the pathogenesis of DN (Sun et al., 2013). Currently, more than 2/3rd of world’s diabetics are dependent on plant based medicine for their successive therapy due to its lesser side effects and low coast (WHO, 2008). Paederia foetida Linn. (Family: Rubiaceae) is a wildly grown woody climber of north-east India. The leaves of the plant are taken as infusion to treat DM by the tribes of Sikkim and Darjeeling Himalayan region. The therapy is given usually for a period 2-3 weeks (Chanda et al., 2013; Chhetri et al., 2005). The vernacular names of the plant include Bhedai lata (Assamese), Gandhabhadulya (Bengali), Gandhali (Hindi), Birilahara (Nepali), Gandhaprasarani (Sanskrit) and Skunkvine or Chinese fever vine (English). The plant is found in central and eastern Himalayas, Malaysia, Philippines, Malacca and India. In India, it is found mainly in Assam, Bengal, Bihar, Meghalaya, Orissa and Sikkim (Chanda et al., 2013; De et al., 1994; Fazlin et al., 2002). The aerial parts of the plant are very rich in paederine, paederenine, paederone, paederolone and iridoid glycosides: asperuloside, scandoside and paederoside. An enzyme present in it, responsible for splitting paederoside and gives its characteristics bad odour when plant is bruised (Shukla et al., 1976; Sugumaran et al., 2005). The plant is also a rich source of friedelin, ursolic acid, ellagic acid, vitamin C, carotene, lupeol, palmitic acid and methyl mercaptan (Soni et al., 2013). In traditional medicine the plant is indicated for the treatment of rheumatism (Charak, 1949), difficulty in labour (Pandey, 1952), vata disorders, piles, oedema and night blindness (Bhattacharya and Bhattacharya, 1933; Dutta, 1902; Krishna-Shastri, 1936). Leaf extract of the plant is reported to possess significant antihyperglycaemic (Kumar et al., 2014; Morshed et al., 2012), antioxidant (Kumar et al., 2014; Osman et al., 2009) and lipid lowering effects (Kumar et al., 2014). A very recent study revealed that the plant effectively suppress NF-kB in animal model (Kumar et al., 2015). Based on above findings, we hypothesized that if Paederia foetida (P. foetida) extract can inhibit NF-kB and hyperglycaemia in vivo, then ALX induced diabetic rat with nephropathy to investigate whether alcoholic leaf extract of the plant affect the NF-kB mediated diabetic renoinflammatory cascades.

2. Materials and method

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2.1 Drugs and Chemicals Glibenclamide (Cat. no. D3525) and Alloxan monohydrate (Cat. no. A7413) were purchased from Sigma Aldrich, St. Louis, MO, USA; and Methanol (Cat. no. 106009) from Merck, Darmstadt, Germany. Carboxymethylcellulose Sodium (Cat.no. C9481) Sodium citrate (Cat. no. PHR1416), Citric acid (Cat. no. C2219000), Phosphate buffer (Cat. no. P5244), Sodium chloride (Cat. no. 746398), Ellman’s reagent (Cat. no. D8130), Thiobarbituric acid (T5500), Lowry reagent (Cat.no. L3540), SOD estimation kit (Cat. no. 19160), Catalase estimation kit (Cat. no. CAT100) and Glutathione (Cat. no. Y0000517) were procured from Sigma Aldrich Corporation. Vettest strips were purchased from IDEXX laboratories, Westbrook, USA. ELISA kits for estimation of pro-inflammatory cytokines and NF-kB were purchased from Life technologies, CA, USA, (TNF-α, catalog no. KRC3011) and Elabscience Biotechnology, Wuhan, China (IL-1β; Cat. no. E-EL-R0012, IL-6; Cat. no. E-EL-R0015 and NF-kB, Cat.no. E-EL-R0674). 2.2 Plant materials Paederia foetida Linn. was collected from Sivasagar district, Assam, during the month of August-September and authenticated in Govt. Ayurvedic college, Guwahati, India. A voucher specimen (no. NIPER/PCT/PTH/01) of the same was deposited in the herbarium of Botanical Survey of India. 2.3 Method of extraction The leaves were separated from the other plant materials and dried under shade. The dried leaves were grounded into coarse powder using a mechanical grinder. Then the powders were soaked in methanol for 7-days and filtered through a muslin cloth. The filtrate obtained was evaporated in a rotatory evaporator (IKA RV10, Malaysia) to get methanolic leaf extract of Paederia foetida (MEPF). The recovery was 17.83% w/w (calculated for dried leaves). Further, the extract was freeze dried to remove the residual water content. Then it was suspended in 0.2% w/v carboxymethylcellulose (CMC) to make a uniform suspension and stored in 2-8οC. 2.4 Animals and induction of diabetes Albino rats (wistar strain), sex male, weighing around 160-180gm were procured from College of Veterinary Science (CVSc.), Khanapara, Guwahati and acclimatized to laboratory conditions maintained at (25 ± 2)οC and 12-hours light-dark cycle for two weeks. The rats 4

were provided with normal pellet diet and tap water ad libitum. After two weeks, rats were grouped in such a way that the variations in weight among the experimental groups are minimized. The overnight fasted animals, except the normal control group, were administered with single intraperitoneal (i.p.) dose of alloxan (ALX), 150 mg/kg body weight (b.w.), in 0.1M citrate buffer, pH 4 (Abdul-Hamid and Moustafa, 2014; Ezejiofor et al., 2014). ALX administered rats were allowed to drink 5%w/v glucose water for 24-hours to prevent acute hypoglycaemia. Fasting blood sugar (FBS) level was measured after 7-days using an Accucheck glucometer from Roche diagnostic, India (Belhekar et al., 2013). Rats with blood sugar level ≥ 200 mg/dL were considered diabetic and included for further study. Before starting the experiment, ethical clearance was taken from Institutional Animal Ethical Committee (IAEC), Gauhati Medical College & Hospital, Guwahati, Assam (approval no. MC/05/2015/55). 2.5 Experimental design One non-diabetic control group and four diabetic groups, each comprising of six rats, were included in the study. We selected two doses of MEPF i.e. 250 and 500 mg/kg b.w. based on a published literature for the treatment of diabetic rats (Kumar et al., 2014). On the other hand, Glibenclamide (GLB) was included in the study as standard antidiabetic drug (Kumar et al., 2012). Group I: Non-diabetic rats receiving 0.2% (w/v) aq. solution of CMC/day for 28-days (p.o.); Group II: Diabetic rats receiving no treatment; Group III: Diabetic rats receiving MEPF at 250 mg/kg b.w./day for 28-days (p.o.); Group IV: Diabetic rats receiving MEPF at 500 mg/kg b.w./day for 28-days (p.o.) and Group V: Diabetic rats receiving Glibenclamide at 10 mg/kg b.w./day for 28-days (p.o.) On 28th day of the treatment, the animals were individually isolated in metabolic cages to measure the urine output for 24-hours. After completion of the study, individual blood glucose levels and body weights of the rats were recorded. Overnight fasted rats were anaesthetized and sacrificed by cervical dislocation after collection of blood samples. Serum was separated from blood samples and stored at – 80οC. Both kidneys were collected and processed with ice-cold saline. A part of the kidneys from each animal was stored in neutral buffered formalin (10% v/v) for histological examination. The rest of the kidneys were then

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homogenized in ice-cold 0.1M phosphate buffer solution (pH 7.4). The tissue homogenates were centrifuged at 12000g using a refrigerated centrifuge (Thermo Scientific Fresco 21), at 4οC, for 30 min. Resultant supernatants were collected and stored at – 80οC for estimation of various parameters. 2.5 Assessment of glomerular function and biochemical changes Glomerular function was assessed by measuring serum and urine creatinine levels using a semi auto analyzer (IDEXX Vet Test Analyzer, 8008). The creatinine clearance (CrCl) was calculated using formula; CrCl= [Urine creatinine (µmol L-1)/Serum creatinine (µmol L-1) X Volume of urine (ml/min.)] (Robertshaw et al., 1989). Moreover, blood urea nitrogen (BUN) and albumin levels were measured by an automated analyzer to assess renal dysfunction. Serum bilirubin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), triglycerides (TRIGs) and total cholesterol (TCHOL) were estimated by same method to evaluate the effects of MEPF on biochemical markers. 2.6 Assessment of oxidative stress MDA (Melondialdehyde) contents in the tissue homogenates were estimated by reacting with thiobarbituric acid, as an index of lipid peroxidation (Okhawa et al., 1979). Superoxide dismutase (SOD) and catalase (CAT) activities in the tissue samples were measured by using commercially available colorimetric assay kits (Sigma Aldrich, St. Louis, MO, USA). Manufacturer’s instructions were followed while carrying out the assays. Quantitation of kidney tissue total protein contents were done by standard Lowry’s method (Lowry et al., 1951). Ellman’s reagent based assay was carried out to determine the activity of GSH in the renal tissue (Ellman, 1959). 2.7 Assessment of diabetic renal inflammation ELISA was carried out to ascertain the effect of MEPF on NF-kB dependent diabetic renal inflammatory pathway. NF-kB p65 ELISA kit was purchased from Elabscience Biotechnology, Wuhan, China. Manufacturer’s protocols were strictly followed while carrying out the assay. Commercially available TNF-α ELISA kit was purchased from Life technologies, CA, USA; and IL-1β and IL-6 ELISA kits were purchased from Elabscience Biotechnology, Wuhan, China. Serum TNF-α, IL-1β, IL-6 and tissue NF-kB levels were estimated out from the standard plots constructed from the absorbance readings of serially diluted standards. 6

2.8 Histopathological assessment After 10 days, formalin stored kidneys were processed consecutively for dehydration and clearing using acetone and xylene. The dehydrated tissues were impregnated into paraffin blocks and cut into thin (5μm) sections, using a rotary microtome (Thermo Scientific, HM 325). The sections were stained with eosin and haematoxylin. Pathological abnormalities in stained sections were identified by a pathologist under 40X magnification without prior information about the groups. 2.9 Statistical analysis One-way analysis of variance (ANOVA) was carried out using Graph-pad Prism software followed by Tukey’s multiple comparison test. Values were expressed in Mean ± SEM; where n=6. Data were considered statistically significant while p<0.05.

3. Results 3.1 Effects of MEPF body weight and blood glucose level Diabetic rats showed significant reduction in their body weight when compared with control rats receiving 0.2% w/v of CMC only. But this was not significantly affected by any of the used doses of MEPF. Diabetic group receiving only ALX, exhibited marked elevation of blood glucose level. MEPF treatment at 500 mg/kg b.w., remarkably restored blood sugar levels in the treated diabetic rats and the effect was comparable to standard drug GLB (Table 1). 3.2 Effects of MEPF on biochemical parameters and renal function Creatinine clearance, an index of GFR, significantly reduced in the diabetic untreated rats. A significant improvement was observed while treated with MEPF; especially at the dose of 500 mg/kg b.w. Diabetic control rats exhibited marked elevation of serum bilirubin, BUN, AST and ALT; whereas serum albumin content significantly decreased. Although, MEPF oral administration reversed these changes, the effects were more pronounced only at the dose of 500 mg/kg b.w. On the other hand, with respect to plasma lipid contents (TRIGs & TCHOL), MEPF in both 250 and 500 mg/kg b.w. doses; significantly normalized its level. The standard drug GLB showed overall significant results (Table 1).

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3.3 Effects of MEPF on antioxidant status MEPF oral administration significantly lowered lipid peroxidation in the treated ALX induced diabetic rat kidney. Renal tissue SOD and CAT of diabetic control group showed significant decline in their activity compared to control group. On MEPF supplementation at a dose of 500 mg/kg b.w., significantly improved activity of these antioxidant enzymes (SOD & CAT). Non-enzymatic antioxidant, GSH also showed significant drop in its activity in the diabetic kidney without treatment. A marked augmented activity of GSH was found in MEPF treated groups. The antioxidant effects of MEPF observed at a dose of 500 mg/kg b.w. on the diabetic rats were comparable to GLB (Table 2). 3.4 Effect of MEPF on diabetic renal inflammation Fig. 2 explains the effects of MEPF on serum pro-inflammatory cytokines (TNF-α, IL-6 & IL-1β) and the renal NF-kB p65 concentrations. The ALX treated diabetic control rat serum showed high concentrations of pro-inflammatory cytokines [Fig. (i) TNF-α, (ii) IL-6 & (iii) IL-1β], whereas a higher expression of NF-kB p65 fraction was detected in the kidney tissue [Fig. (iv)]. MEPF treatment significantly supressed these serum pro-inflammatory cytokines; but at a dose of 250 mg/kg, failed to produce significant alteration of serum IL-6 level. Renal tissue NF-kB p65 fraction was dose dependently down regulated by 28-days oral MEPF treatment. 3.5 Effect of MEPF on renal histopathology Fig. 3 depicts the comparative diabetic renal pathology in the presence or absence of MEPF and GLB; under microscope (40X magnification). Fig. 3(i) represents renal microscopic view of a nondiabetic control rat (black arrows), showing normal glomerular architecture ( proximal convoluted tubules (

) and distal convoluted tubules (

),

); Fig. 3(ii) illustrates a

untreated diabetic rat kidney section with complete loss of nomal glomerular architecture showing detachment of basement membrane, glomerular endothelial cell damages, glomerular space reduction, tubular damage with necrosis (red arrows); Fig 3(iii) displays a diabetic kidney section on 250 mg/kg b.w. MEPF treatment with mild improvement in glomerular appearance and tubular structure (blue arrows); Fig 3(iv) represents a diabetic kindney on MEPF 500 mg/kg b.w. treatment with greatly improved glomerular and tubular structure, healed nectrotised tubules, restored glomerular spaces in comparison with lone ALX treated diabetic kidney (blue arrows). Fig. 3(v) shows diabetic kidney section on 10 mg/kg GLB treatment with almost restored normal renal architecture (green arrows). 8

4. Discussion The pathogenesis of DN has been related to both oxidative stress and chronic renal inflammation. However, source of oxidative stress producing oxygen derivatives (i.e. reactive oxygen species, ROS) in the kidney is not clearly defined.

Studies on human DN have

linked ROS in renal cells to high blood glucose concentrations in diabetic individuals (Brownlee, 2005; Susztak, 2006). On the other hand, lipophilic derivatives of ALX can also cause selective toxicity to GLUT2 expressing renal tubular cells. However, the toxicity is severe only on systemic administration (Lenzen, 2008). Likewise, renal inflammation in human DN is reported to be NF-kB linked and pro-inflammatory cytokines can initiate the event (Balakumar et al., 2009; Mezzano et al., 2004). Plant originated drugs, in terms of safety profile, always find advantages over existing synthetic drugs for the management of diabetes and its complications. Here, we investigated the effect of indigenous plant P. foetida extract, a folk antidiabetic medicine (Chhetri et al., 2005), recently reported to have inhibitory action against transcription factor, NF-kB (Kumar et al., 2015), on various oxidative stress markers and NF-kB dependent renal inflammatory pathway of DN. During the experimental period, the ALX treated diabetic rats exhibited hyperglycaemia, polydipsia and polyuria; the typical characteristics of DM (American Diabetes Association, 2010). At end of the experiment, on serum analysis, we found that the serum bilirubin, BUN and creatinine levels in the diabetic control rats were considerably higher than that of nondiabetic control rats. On the other hand, serum albumin and GFR decreased to a greater extent. These findings confirmed glomerular injury and renal dysfunction in the ALX treated groups (Thomas and Karalliedde, 2015). We observed a significant reversal of altered serum markers and GFR in the MEPF treated groups, after 28-days oral administration. However, the effects were more pronounced at a dose of 500 mg/kg b.w. Also, at 500 mg/kg b.w. dose, MEPF remarkably normalized the elevated blood sugar level in the treated diabetic rats. Our findings were consistent with the previous reports of a reduction in blood glucose level in rats with DM by P. foetida extract (Ahmed et al., 2014; Morshed et al., 2012). Blood glucose lowering property of the extract could be due to the presence of iridodial glycoside contents (i.e. scandoside) and ursolic acid in it (Miura et al., 1996; Zhang et al., 2006). Ursolic acid facilitates phosphorylation of insulin receptors and stimulates glucose uptake through 9

inhibition of protein tyrosine phosphatase 1B (Zhang et al., 2006). Moreover, it can potentiate the beta cell function in a partially damaged pancreatic islet (Jang et al., 2009). In DM, metabolic and regulatory processes get altered due to lack of insulin availability precipitating hyperlipidemic state. Lipids such as TRIGs and TCHOL start accumulating in various body parts. The veins and arteries are more prone to such attacks leading to vascular complications of DM; related to lipid deposition (Goldberg, 1981). In the present study, we observed that serum TRIGs and TCHOL levels in hyperglycaemic, diabetic rats were abnormally high. Oral treatment with MEPF significantly normalized this elevated serum lipids. Three bioactive principles viz. asperuloside, ellagic acid and ursolic acid, present in it, could be responsible for the observed antidyslipidemic action (Hirata et al., 2011; Kannan and Quine, 2013; Somova et al., 2003). A study on asperuloside clearly demonstrated that this glycoside, in hyperlidemic state, can stimulate metabolic activities across several organ systems (Fujikawa et al., 2012). Alike, hyperlipidemia reducing ability of ellagic acid has been linked to HMGCoA reductase, the key enzyme responsible for cholesterol biosynthesis, inhibiting property (Kannan and Quine, 2013). In DN, lipid peroxidation increases with the substantial failure of antioxidant machineries (SOD, CAT and GSH) in the kidney tissues leading to oxidative stress. Decreased activity of SOD, CAT and GSH in diabetes; protecting cell membrane and its constituents from oxidative damage, is due to over production of ROS (Giacco and Brownlee, 2010). In the present study, the decreased activity of SOD, CAT and GSH, in the kidneys of the diabetic rats with no treatment, indicated oxidative stress. Oral treatment with MEPF revitalized the diminished activity of these enzymatic (i.e. SOD and CAT) and non-enzymatic (i.e. GSH) free radical scavengers. Recently, potentiating of bodily free radical scavenging system activity by P.foetida leaf extract has been reported, supporting the present findings (Kumar et al., 2014). Moreover, we observed a simultaneous increase in the level of thiobarbituric acid reactive substances (MDA) along with the decrease in activity of free radical scavenging system in the kidney of control diabetic rats. This confirmed a high degree of

lipid

peroxidation in the ALX treated diabetic rats receiving no treatment (Chaudhry et al., 2007; Prince et al., 1998). MEPF administration greatly reversed this increased renal tissue MDA level. This reversal could be due to the presence of bodily free radical scavenger sparing principles like ellagic acid, friedelin, ursolic acid etc in the extract (Somova et al., 2003; Sunil et al., 2013; Yüce et al., 2008). The free radical scavenging activity and anti-lipid

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peroxidation property of ellagic acid has been attributed to its metal chelating action (Yüce et al., 2008). A study on human DM detected higher concentrations of TNF-α than the normal, in the patient’s serum, especially in those with DN (Moriwaki et al., 2003). The pathogenetic potential of this pleiotropic cytokine is because of its ability to cause direct renal cell injury. Moreover, it activates secondary messenger system, cell adhesion molecules, transcription factors, synthesis of cytokines, acute phase proteins, MHC molecules etc. (Navarro-González et al., 2009). In this study, we detected abnormally elevated serum level of TNF-α in the untreated hyperglycaemic diabetic rats. On MEPF oral administration, we found a dose dependent inhibition of this cytokine in the treated diabetic groups. We also detected an elevated serum IL-6 concentration in control diabetic rats. This was in line with a previous report on diabetic patients where serum analysis of patients with nephropathy detected markedly elevated IL-6 concentrations (Dalla Vestra et al., 2005). Investigations on DN, demonstrated that IL-6 is involved in glomerular basement membrane thickening, alterations in endothelial permeability and induction of mesangial cell proliferation (Mora and Navarro, 2006). MEPF administration, at a dose of 500 mg/kg, led to pronounced decrease in serum IL-6 level. But at a dose of 250 mg/kg, failed to effect significantly. A study on IL-1β involvement pattern in DN reported that the circulating serum IL-1β level in albuminuric patients is remarkably high and its level increases in the renal cortex of db/db mice in an age dependent fashion (Shahzad et al., 2015). We indeed observed a high serum IL-1β level in the diabetic untreated group with reduced serum albumin. IL-1β is thought to be associated with abnormal intraglomerular hemodynamic changes. Additionally, it has been found to cause increase in vascular endothelial permeability and induction mesangial cell proliferation and matrix synthesis (Mora and Navarro, 2006). In contrast to diabetic control rats, MEPF treated rats exhibited a significant downturn in serum IL-1β level. The mechanistic events of cytokine inhibition by MEPF are difficult to predict at present. It could be due to its hyperglycaemia lowering property in diabetes. Renal inflammation in DM and activation of transcription factor, NF-kB; both are positively co-related (Balakumar et al., 2009). Pro-inflammatory cytokines can act as stimuli for NF-kB activation; notably including IL-1β and TNF-α (Sanz, 2010). Activation leads to the degradation of inhibitory IĸB fraction by phosphorylating p65 subunit which allows p65 subunit to translocate into the nucleus. On binding to the target site on DNA, active NF-kB promotes transcription of several downstream genes directly or indirectly associated DN 11

(Mezzano et al., 2004; Sanchez and Sharma, 2009). Moreover, it promotes the transcription of pro-inflammatory cytokines; including TNF-α and IL-1β (Sanz, 2010). More advanced studies on human diabetic nephropathy reported that NF-kB highly upregulated in diabetic kidney. (Mezzano et al., 2004). Here we detected remarkably high concentration of p65 subunit in the untreated diabetic kidney through ELISA while in MEPF treated diabetic groups the active NF-kB concentrations were significantly low. Supporting our findings, a recent study demonstrated that P. foetida extract can efficiently inhibit transcription factor NF-kB activation to suppress inflammatory response (Kumar et al., 2015). This property MEPF may possibly be due to synergistic effect of ursolic acid, lupeol and ellagic acid present in it (Anitha et al., 2013; Lee et al., 2016; Shishodia et al., 2003). However, MEPF is comparable to standard antidiabetic GLB in terms of protective actions against early stage diabetic nephropathy. The above results are in support of our hypothetical mechanism of renoprotection in diabetes by MEPF (illustrated in Fig. 4). Further, in renal histopathology of the diabetic control group, we observed oxidative glomerular damages, chronic inflammatory changes and intrinsic cells undergoing necrosis. In the kidney sections of GLB treated diabetic group, a slight distortion in renal microarchitecture was observed with majority of changes in its tubular structures. On investigations of MEPF treated kidney sections, we saw some minor improvements at a dose of 250 mg/kg, whilst at a dose of 500 mg/kg rats exhibited renal microarchitectures with healed tubular cells, slightly distorted glomerulus and preserved glomerular spaces. The observed renoprotection of the extract could be due to suppression of hyperglycaemia mediated oxidative stress and inhibition of renal inflammatory cascades associated with NFkB activation.

5. Conclusion In conclusion, P. foetida methanolic leaf extract exerted prominent nephro-protection in early stage of experimental DN in rats. Our study reports that the renoprotective action is due to attenuation of renal oxidative stress and suppression of renoinflammatory events though inhibition of NF-kB, in early progressive DN. Thus, it might be useful during the early stage of DN. However, our study further necessitates in detail investigation on P. foetida to find out the active principles responsible for its nephroprotective action in diabetes.

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Conflicts of interest Authors declare there is no conflict of interest in respect of this study.

Authors contributions Dr. Mangala Lahkar ([email protected]) provided overall guidance while designing the experiment,

model

establishment

and

manuscript

writing.

M.

P.

Borgohain

([email protected]) contributed to the experimental design, model establishment and manuscript writing. K. Devasani ([email protected]) contributed to the manuscript writing. T.J. Das ([email protected]) collected the plant materials and performed the necessary

works

for

authentication.

T.

J.

Das

and

A.

Mohapatra

([email protected]) carried out animal dosing and sample collection. L. Chowdhury

([email protected])

and

S.

Ahmed

([email protected]) carried out biochemical estimations and ELISA. N. Bolshette ([email protected]) performed statistical analysis.

Acknowledgement This study was financially supported by National Institute of Pharmaceutical Education and Research, Ministry of Chemical and Fertilizers, Govt. of India. The authors are thankful to IBT-hub NIPER Guwahati and Gauhati Medical College, Guwahati, Assam for providing facilities to carry out the research work.

Reference Abdul-Hamid, M. and Moustafa, N., 2014. Amelioration of alloxan-induced diabetic keratopathy by beta-carotene. Experimental and Toxicologic Pathology 66, 49-59. Ahmed, A.M.A., Islam, M.M., Rahman, M.A. and Hossain, M.A., 2014. Thrombolytic, cytotoxic and antidiabetic effects of Paederia foetida L. leaf extract. British Journal of Medicine and Medical Research 4, 1244. American Diabetes Association, 2010. Diagnosis and classification of diabetes mellitus. Diabetes care 33, S62-S69.

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Fig. 1 Experimental design of the study. Fig. 2 Effects of MEPF on pro-inflammatory cytokines TNF-α (i), IL-6 (ii), IL-1β (iii), and NF-kB p65 fraction (iv). Values are expressed in Mean ± SEM (where n=6). One-way ANOVA was carried out followed by Tuky’s multiple comparison test. Values are considered statistically significant when p< 0.05 and where ap< 0.05 when compared with Normal control; bp< 0.05 when compared to diabetic control; ns non-significant when compared with diabetic control; ns* non-significant when compared to normal control. Fig 3: Photomicrograph of Haematoxylin and Eosin stained section of Kidney. (i) Control, (ii) Diabetic rat, (iii) Diabetic+MEPF250, (iv) Diabetic+MEPF500 and (v) GLB10. Fig. 3(i) renal microscopic view of a control rat (black arrows), showing normal glomerular architecture (

), proximal convoluted tubules (

) and distal convoluted tubules (

).

Fig. 3(ii) diabetic kidney section, with complete loss of nomal glomerular architecture 18

showing detachment of basement membrane, glomerular endothelial cell damages, glomerular space reduction, tubular damage with necrosis (red arrows); Fig 3(iii) diabetic kidney section on MEPF 250 mg/kg, with mild improvement in glomerular appearance and tubular structure (blue arrows); Fig 3(iv) diabetic kindney on MEPF 500 mg/kg, with greatly improved glomerular and tubular structure, healed nectrotised tubules, restored glomerular spaces (blue arrows). Fig. 3(v) diabetic kidney section on 10 mg/kg GLB, with almost restored normal renal architecture (green arrows). Fig 4: Diagrammatic representation showing possible mechanism of MEPF on oxidative stress and inflammatory pathway of DN. MEPF supresses the oxidative stress exerted by ALX and inhibit NF-kB mediated production of pro-inflammatory cytokines.

Table 1 Effect of MEPF on biochemical parameters in ALX induced diabetic rats. Parameters

Control

Diabetic Control

Diabetic + MEPF (250mg/kg)

Diabetic + MEPF (500mg/kg)

Diabetic+ GLB (10mg/kg)

Body Weight

368.69 ±

182.22 ±

210.54 ±

224.27 ±

272.65 ±

(gm.)

11.04

6.98a

6.70ns

14.66ns

9.20b

Blood Glucose

92.33 ±

489.83 ±

402.33 ±

349.17 ±

103.00 ±

a

ns

b

(mg/dL)

8.27

27.08

33.32

31.18

16.42b

BUN (mg/dL)

7.55 ±

15.72 ±

13.92 ± 0.82ns

11.82 ± 0.53b

8.84 ± 0.43b

0.71

0.81a

Creatinine

42.63 ±

64.93 ±

52.27 ± 2.08b

49.60 ± 1.10b

44.98 ± 1.98b

(mg/dL)

2.29

3.00a

Creatinine

2.83 ±

0.90 ± 0.43a

1.71 ± 0.08ns

2.36 ± 0.12b

2.64 ± 0.29b

Clearance

0.48 0.91 ± 0.07a

0.82 ± 0.08ns

0.67 ± 0.03b

0.60 ± 0.05b

21.22 ±

35.92± 2.65b

39.45 ± 3.33b

42.50 ± 2.80b

(ml/min) Total Bilirubin

0.28 ±

(mg/dl)

0.03

Albumin (g/L)

43.34 ±

19

AST (IU)

2.12

5.33

43.27 ±

78.50 ±

58.49 ± 6.52ns

a

6.88

7.04

58.04 ±

63.52 ±

0.99

1.03ns*

TCHOL

32.80 ±

158.60 ±

(mg/dL)

3.33

8.59a

TRIG (mg/dL)

53.59 ±

112.42 ±

2.80

10.50a

ALT (IU)

70.25 ± 3.34ns

52.10 ± 3.57b

56.44 ± 2.81ns

49.97 ± 1.76b

31.60 ± 2.02b

116.74 ± 4.50b 98.38 ± 4.91b

81.08 ± 7.50b

86.23 ± 6.04b

69.27 ± 2.79b

79.00 ± 5.42b

Values are expressed in Mean ± SEM (where n=6). One way ANOVA followed by Tuky’s multiple comparison test. Values are considered statistically significant when p< 0.05 and where; ap< 0.05 when compared with Normal control; bp< 0.05 when compared to diabetic control; ns non-significant when compared with diabetic control; ns* non-significant when compared to normal control.

Table 2 Effects of MEPF on oxidative indices and lipid peroxidation. Parameters

Control

Diabetic control

Diabetic + MEPF (250mg/kg)

Diabetic + MEPF (500mg/kg)

Diabetic+ GLB (10mg/kg)

MDA (nmol /mg of protein)

9.72± 1.03

52.50±1.93*

38.34±3.76**

31.72±3.99**

18.39±4.39**

SOD (U/mg

11.10±0.84

2.98 ±0.62*

5.84±0.97

8.07 ±1.06**

9.73±1.32**

CAT (U/mg of protein)

76.61±4.68

28.71±8.9*

41.29±9.21

59.66±4.96**

64.43± 5.41**

GSH

81.09±3.73

55.69 ±2.73*

70.74±

78.28± 3.49**

77.71±3.73**

of protein)

4.09** Values are expressed in Mean ± SEM (where n=6). One way ANOVA followed by Tuky’s multiple comparison test. Values are considered statistically significant when p< 0.05 and where *p< 0.05 when compared with normal control; **p< 0.05 when compared to diabetic control.

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Graphical abstract

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