Background: Diabetes-instigated nerve damage is a chronic complication including impaired peripheral nerve function with lowered nerve conduction velocity (NCV), demyelination of nerve fibres and alterations in the behaviour. Many clinical and experimental studies have proved that Phyllanthus amarus and esculetin have potential effects against various diseases. Purpose: The aim of this study is to assess the neuroprotective role of hydro-ethanolic extract of Phyllanthus amaras (PAE) and esculetin (ESC) on NCV, metabolism, behavioural and structural changes in diabetic rats. Methods: The extent of protection using PAE and ESC in diabetic rats was determined by checking the HbA c, NO, + + myeloperoxidase (MPO), total calcium, protein content, Na -K ATPase activity, acetylcholine content and behavioural alterations using rotarod and maze learning tests on 7, 14 and 21 days. NCV was measured on the 21st day. + + Results: The diabetic rats showed increased HbA1c, nitrite, MPO, calcium and decreased protein, Na -K ATPase activity, NCV, acetylcholine, behavioural alterations and morphological changes of sciatic nerve so that diabetic peripheral neuropathy (DPN) is manifested. Continuous treatment for three weeks with Phyllanthus amarus and esculetin significantly minimized the damage to axons and myelin sheath and enhanced the sciatic NCV by reversing all the mentioned parameters. Conclusion: Phyllanthus amarus and esculetin showed the anti-diabetic as well as anti-inflammatory characteristics by prevention of initiation of adverse processes of the sciatic nerve morphology, internal cell functions leading to improved coordination, behavioural and physiological functions in STZ-induced diabetic rats. PAE has shown similar effects with the ESC. However, further studies are essential to confirm their detailed therapeutic effects. Keywords Neuropathy, diabetes, myelin, peripheral nerve the ischemia and tissue hypoxia which leads to the disruption Introduction of microvasculature. This further causes the endothelial dysfunction and finally segmental demyelination of peripheral Diabetic neuropathy is a syndrome consisting of progressive nerves. Increase in aldose reductase (AR) activity (activation of clinical disorders that affect various parts of the peripheral polyol pathway) indirectly affects the concentration of proteins, nervous system. It is a common, costly and serious complication HbA1C level, abnormal axonal nerve conduction velocity, of diabetes, and risk factor for ulcers and amputations. It occurs cholinergic neurotransmitter synthesis, elevation of ROS, RNS in more than 50 per cent of diabetic mellitus (DM) patients species, microangiopathy and deprivation of fibre density. These but is largely unreported or untreated such that only 8–10 reported manifestations are responsible for the loss of balance per cent cases may be present at diagnosis level. Etiology of and coordination, and impairment of cognitive behaviour leading diabetic neuropathy is complex and multi-factorial which is not to sensory loss and axonal atrophy of myelinated fibres. completely understood. Nerve function impairment is mostly displayed in both experimental and clinical models of diabetes. Department of Zoology, Neuroscience Lab, Osmania University, Hyderabad, Evidence is that cognitive deficit, dementia and degeneration of Telangana, India. neurons prevail in older people of type-2 diabetic patients. There Corresponding author: is a functional deficit on sensory and motor coordination using Karnati Pratap Reddy, Department of Zoology, Neuroscience Lab, Osmania rotarod performance test against peripheral sensory neuropathy. University, Hyderabad, Telangana 500007, India. Chronic hyperglycemia, oxidative and nitrosative stress produce E-mail: email@example.com Creative Commons Non Commercial CC BY-NC: This article is distributed under the terms of the Creative Commons Attribution- NonCommercial 4.0 License (http://www.creativecommons.org/licenses/by-nc/4.0/) which permits non-Commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Open Access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage). 18 Annals of Neurosciences 26(3–4) Currently, the usage of non-synthetic drugs obtained Methods from plant sources has got the attention due to their generous availability and efficacy in decreasing the unfavourable drug Experimental Animals reactions. In the clinical trials, different protective agents were Male wistar rats (200±50g) were procured from NCLAS, used from different classes of drugs such as antidepressents, NIN, Hyderabad, Telangana, India. They were maintained anticonvulsants, AR inhibitors, PKC inhibitors, capsaicin, at an ambient temperature of 22±2 C with a 12 h light/ lidocaine, opiods, α-lipoic acid, benfotiamine (Vit. B1 Analogue), dark cycle. Protocols of the study were duly approved by and natural antioxidants such as curcumin, acetyl-L-carnitine, the Animal Ethics Committee (CPCSEA No.: 383/01/a/ vitamins A, C, and E, melatonin, date extract and α-Lipoic acid 7 CPCSEA), Department of Zoology, Osmania University, which are relatively effective for characteristic relief. Hence, a Hyderabad, Telangana, India, and CPCSEA guidelines were novel approach for slowing down the accurate progression of followed for conducting the studies. The animals were given diabetic peripheral neuropathy (DPN) disease is essential. standard pellet diet and normal water ad libitum all through Phyllanthus amarus is a commonly available weed 8 9 the study. in India. Its anti-hyperglycemic, anti-nociceptive, anti- inflammatory and anti-carcinogenic properties were attributed 10,11 due to its extensive anti-oxidant effect. Besides, it exhibits Drugs and Chemicals kinetic inhibition on carbohydrate-metabolizing enzymes due 12 13 to the presence of polyphenolic content, affinity for AR STZ and esculetin were purchased from Sigma Aldrich, and can regenerate the sciatic nerve crush injury. Three India. Hydro-ethanolic PAE was prepared in our lab and the pure pentacyclic triterpenoids—oleanolic acid, ursolic acid dose level of Phyllanthus amarus and esculetin were selected and lupeol—present in aerial parts were able to inhibit the from the previous report and described previously. Further α-amylase activity. Polyphenols and vitamin C present in chemicals and reagents used were of analytical grade in this extract of Phyllanthus amarus (PAE) exhibit pronounced experiment. systemic antinociceptive effect, particularly in experimental models of neuropathic pain. Aerial parts of PAE containing alkaloids, saponins, lignans, flavonoids, galloatinoids, Induction of Experimental Diabetes terpinoids and glycosides are known to reduce hyperglycemic After overnight fasting, the rats were induced diabetic levels in streptozotocin (STZ)-induced diabetic rats and were condition with STZ (60 mg/kg b.w, i.p in sodium citrate buffer responsible for its pleotropic properties against various diseases. (0.1 M) at pH 4.5). Diabetic state was confirmed after 48 h Hydro-ethanolic PAE and a coumarin derivative ‘esculetin’ by determining glucose levels from tail vein blood using test (bioactive compound), able to control hyperglycemia of blood strips (Dr. Morepen Gluco One). Animals having a glucose and reversal of anti-oxidant status, and reduce the rate of level of more than 250 mg/dL in blood were considered for progression of diabetic neuropathy in STZ-induced diabetic the present study. rats, were reported previously in our lab. Esculetin, a potent antioxidant is associated with the health 19,20 benefits by consuming fruits and vegetables. Previous study on Experimental Design esculetin has reported the inhibition of BChE, AChE and BACE1 enzymes and showed the protective effect against Alzheimer’s The animals were separated into six groups, each disease. It has a systemic inhibitory effect on hyperglycemia containing six rats. Group 1: Normal control (NC). Group by regulating the enzymes of carbohydrate metabolism, AR in 2: Diabetic control (DC)—received STZ (60 mg/kg b.w, vitro and cataractogenesis, which has a broad range of usage in i.p), observed for 24 days. Group 3: D+PAE-Diabetic rats the emerging drugs. It has an anti-proliferative activity against treated with ethanolic PAE (400 mg/kg b.w/ml/day p.o) hepatocellular carcinoma and proved its anti-nociceptive effect for 21 days (day 3–day 24). Group 4: D+ESC-Diabetic on inflammatory and non-inflammatory rat models. It showed rats treated with the esculetin (45 mg/kg b.w/ml/day i.p) antioxidant effect by impeding 3T3-L1 cellular adipogenesis for 21 days (day 3–day 24). Group 5 and Group 6: Rats and has neuroprotective activity on degenerated axons or received only PAE and only esculetin, respectively (day dendrites of brain cells. It has shown its extensive therapeutic 3–day 24) which are non-diabetic rats. After PAE and effect on various diseases. Esculetin can preserve the COX and ESC treatment, day 7, 14 and 21 rats were subjected to GSH/GSSG ratio in mice model of stress and unabled contextual motor coordination test and maze learning test to assess memory. the developed neuropathy. Nerve conduction velocity In the present study, hydro-ethanolic PAE and, its was determined on the 21st day and the blood was one of the secondary metabolite derivative, esculetin (6, collected retro-orbitally for the estimation of glycosylated 7-dihydroxy coumarin) were tested further on functional haemoglobin (HbA1c) level. Day 7, 14 and 21 rats were recovery, structural changes and behaviour of rats against sacrificed and sciatic nerves were taken out for further diabetic neuropathy to know their neuro-potential benefits. biochemical and histopathological studies. Srilatha and Reddy 19 Abbreviations: NC, normal control; DC, diabetic control; D, diabetic; PAE, Phyllanthus amarus extract; ESC, esculetin; STZ, streptozotocin. Rotarod (Motor Coordination) Test Estimation of Glycosylated Hb The rotarod (Dolphin TM instruments) test measures the Procedure given by Nayak and Pattabiraman was followed riding time (in seconds) staying on the rotating rod by for the determination of glycosylated HbA1c. To the avoiding falling on the ground. Animal’s stay time on the mixture of hemolysate, normal saline and 1 ml of oxalic rotating rod gives an indication of the level of balance, fore acid, 1 ml of TCA reagent was added. After centrifugation, and hind limb coordination, physical condition and motor to the obtained supernatant, TBA reagent was added and activity (innate antagonistic reflex activity). Initial time of the then optical density was measured at 443 nm to calculate instrument was set to 0 seconds and the rotating speed was the percentage of GHb. to 30 rpm (constant mode) prior to the experiment. Before experimental testing, rats were trained for three consecutive Estimation of Nitrite/In Vivo Antioxidant Activity days by keeping the animal on the rotating rod for 2 minutes. Performance of the each rat was measured as the maximal Nitrite accumulated in the supernatant is the measure of time spent on the rod before falling off and endurance time nitric oxide (NO) produced and was determined according was noted in seconds. to the method of Green et al. Using Greiss reagent, nitrite concentration was studied at 546 nm on spectrophotometer and the amount of nitrite was expressed as µg/mg of protein. Maze Learning Test Maze learning is the practice of learning by rats over a Myeloperoxidase Estimation/Anti-inflammatory Activity complex branching passage, which appears like a puzzle. The overnight fasting animals trying to acquire the food/target is Myeloperoxidase (MPO) was assessed by the method of the primary method of observing spatial learning which is Bradley et al. One unit of MPO activity was expressed in popularly used in behavioural laboratory. Before starting the terms of the amount of MPO degrading one micromole of experimentation, the rats from all groups were trained for two peroxide at 25 C per 1 min (U/g tissue) by measuring the 32,33 days and the time to reach the food was calculated. absorbance at 460 nm. 20 Annals of Neurosciences 26(3–4) for about 24 hours followed by fixation in 1 per cent osmium Estimation of Calcium tetraoxide, then dehydrated in an increased concentration of Total amount of calcium in sciatic nerve was estimated as a series of graded alcohols. These samples were permeated, detailed by Severinghaus and Ferrebee with modification embedded with araldite resin and then incubated for 72 hours 37,38,39 as suggested (Muthuraman et al.). The Ca levels are for proper polymerization at 80 C. Using a glass knife, 60 nm expressed in ppm/mg of protein. ultra-thin sections were mould on microtome (Leica Ultra cut UCT-GA-D/E-1/00) and then mounted over the copper grids. Staining was done using saturated aqueous uranyl acetate and Protein Estimation counter staining was done using Reynolds lead citrate and Amount of protein available in the sciatic nerve tissue was then observed under transmission electron microscope (H- estimated as reported by Lowry et al. 7500, HITACHI, Japan). + + Na -K ATPase Statistical Analysis + + Na -K ATPase enzyme activity in the sciatic nerve was Data obtained was subjected to one-way analysis of variance measured according to Kaplay and liberated inorganic Pi (ANOVA) to compare the dissimilarities between and within was determined by Taussky and Shorr. The activity was + + the groups. The results were presented as Mean ± SEM. The indicated as micromoles of Pi liberated/hr/g of Na -K ATPase assessment of mean values was done using Student’s t-test at enzyme in sciatic nerve tissue of rat. a significance of p < 0.05. Nerve Conduction Velocity Measurements After anesthetizing the rats with sodium pentabarbitone (60 Results mg/kg b.w. i.p) from each group, sciatic nerve was exposed and bipolar needle electrodes (PowerLab, AD instrument, Changes in Motor Coordination, Maze Learning Model: ML825, Serial: 225-0907, Australia, frequency: 20 Abilities Hz, amplitude: 1.5 V and duration: 0.1 ms) were placed at two sides of sciatic nerve. Rectal temperature continued The behavioural studies using rotarod and maze learning (37 C) with a warm blanket tissue, drying was avoided were conducted on day 7, day 14 and day 21 rats of all the by adding mammalian ringer solution. Evoked potential groups. Streprozotocin-affected diabetic rats have produced response (m/s) was calculated by dividing the distance (mm) a significant decrease (34.68%, 47.82% and 53.78% on and the difference in time (ms) between two stimulated day 7, 14 and 21, respectively, p < 0.001) in time latency points. on rotarod at 30 rpm and so affected the motor coordination of rats. Treatment of diabetic rats with Phyllanthus amarus extract (–17.47%, –47.44% and –82.21% on day 7, 14 and Acetyl Choline Estimation 21, respectively, p < 0.01) and esculetin (-22.49%, -51.63%, Estimation of acetyl choline was carried according to the -91.47% on day 7, 14 and 21 respectively, p<0.005) for Hestrin method. The content of acetylcholine was expressed three weeks produced a significant redemption on motor as mg of Ach/gm weight of sciatic nerve tissue. coordination (Figure 1). The diabetic rats exhibited much longer time (–67.69%, –108.74% and –203.85% on day 7, 14 and 21, respectively, p<0.001) to reach the goal point in the Light Microscopy maze setup than the control rats (Figure 2). Increase of the The rats were subjected to anesthetization using 10 per cent latency period to achieve the goal and significant decrease chloral hydrate (350 mg/kg b.w, i.p). After cleaning the skin of learning ability on maze was observed in diabetic rats using 70 per cent alcohol, vertical incision was made along compared to the control rats. The diabetic rats treated the thigh; sciatic nerves were exposed and excised rapidly. with PAE (11.26%, 17.18% and 37.86% on day 7, 14 and They were transversely sectioned, processed independently, 21, respectively, p < 0.01) and ESC (14.56%, 23.43% and and stained using haematoxylin and eosin (H&E) stain. 44.72% on day 7, 14 and 21, respectively, p < 0.01) showed Then the sections were examined under light microscope decrease of the latency period to achieve the goal leading to (NLCD-307B, Lawrence Mayo, China). the recovery of learning ability significantly in contrast to the diabetic rats. The esculetin-treated diabetic rats spent more time on rotarod and reached the goal faster on maze than Transmission Electron Microscopy (TEM) PAE-treated diabetic rats. The results obtained by rats that The another part of sciatic nerves were fixed using 2.5 per cent received only PAE and ESC groups were similar to those of glutaraldehyde (0.1 M phosphate buffer, pH 7.2), kept at 4 C control group. Srilatha and Reddy 21 Figure 1. Effect of Phyllanthus amarus Extract and Esculetin on Mo- Figure 2. Effect of Phyllanthus amarus Extract and Esculetin on tor-Coordination on Rotarod (Sec) in STZ-Induced Diabetic Rats Maze Learning Behaviour in STZ Induced Diabetic Rats (Min) Abbreviations: NC, normal control; DC, diabetic control; D, diabetic; PAE, Abbreviations: NC, normal control; DC, diabetic control; D, diabetic; PAE, Phyllanthus amarus extract; ESC, esculetin; STZ, streptozotocin. Phyllanthus amarus extract; ESC, esculetin; STZ, streptozotocin. Note: The signiﬁcant differences in between the groups were compared using Note: The signiﬁcant differences in between the groups were compared one-way ANOVA followed by t-test. Data presented as Mean ± SEM (n = 6). using one-way ANOVA followed by t-test. Data presented as Mean ± SEM (n = 6). P < 0.001 against normal group and *p < 0.01, **p < 0.005 against diabetic group. Table 1. Effects of Phyllanthus amarus Extract and Esculetin on Glycosylated Haemoglobin (%) in STZ-Induced Diabetic Rats % Change % Change % Change % Change % Change from from from from from Groups NC DC Control D+PAE Control D+ESC Control PAE Control ESC Control HbA1c 4.78± 8.31± –73.84 5.87± –22.80 6.23± –0.42 4.80± –0.41 4.88± –0.20 # * ** (%) 0.33 0.21 0.17 0.36 0.34 0.35 Abbreviations: NC, normal control; DC, diabetic control; D, diabetic; PAE, Phyllanthus amarus extract; ESC, esculetin; STZ, streptozotocin. Note: The signiﬁcant differences in between the groups were compared using one-way ANOVA followed by t-test. Data presented as Mean ± SEM (n = 6). P < 0.001 against normal group and *p, **p < 0.001 against diabetic group. Table 2. Effects of Phyllanthus amarus Extract and Esculetin on Myeloperoxidase (MPO) Activity (µmol of Peroxide/min/gm Tissue) in Sci- atic Nerve Tissue of STZ-Induced Diabetic Rats % Change % Change % Change % Change % Change from from from from from Groups NC DC Control D+PAE Control D+ESC Control PAE Control ESC Control MPO 17.50± 90.17± –415.25 58±2.67 –231.42 47.33± –170.45 17.83± –1.88 18± –2.85 # ** 1.12 8.31 1.36 1.19 1.06 Abbreviations: NC, normal control; DC, diabetic control; D, diabetic; PAE, Phyllanthus amarus extract; ESC, esculetin; STZ, streptozotocin. Note: The signiﬁcant differences in between the groups were compared using one-way ANOVA followed by t-test. Data presented as Mean ± SEM (n = 6). P < 0.001 against normal group and *p, **p < 0.01 against diabetic group. Changes in Glycosylated Haemoglobin Myeloperoxidase Activity Glycosylated haemoglobin (HbA1c) was almost doubled in MPO activity increased significantly in DPN rats when STZ rats (Table 1). After three days, polydipsia, polyuria and compared to the control rats (Table 2). Treatment with polyphagia were seen in the diabetic rats. HbA1c was decreased esculetin decreased the MPO levels more significantly. significantly when treated with Phyllanthus amarus as well as PAE has shown the lowered MPO levels as much as that esculetin and maintained the glycosylated haemoglobin near to of esculetin-treated group which maybe due to extensive the normal range. This proves the role of Phyllanthus amarus phytoconstituents present in the plant extract. and esculetin in controlling the blood glucose. 22 Annals of Neurosciences 26(3–4) Figure 4. Effect of Phyllanthus amarus Extract and Esculetin on Pro- Nitrite Content tein Content in Sciatic Nerve Tissue (mg/gm Weight of Tissue) of Total amount of NO was significantly increased in diabetic STZ-Induced Diabetic Rats control rats (–127.20%, –129.92 and –135.29 on day 7, 14 and 21, respectively, p < 0.001). Treatment with PAE (11.97%, 22.53% and 37.18% on day 7, 14 and 21, respectively, p < 0.05) and ESC (14.88%, 34.06% and 44.37% on day 7, 14 and 21, respectively, p < 0.05) attenuated the elevated NO levels (Figure 3). Calcium Content A significant increase in the total calcium level was observed in the sciatic nerve tissue of diabetic rats in contrast to the normal group (Table 3). The diabetic rats received with PAE (400mg/kg, p.o) and ESC (45mg/kg, i.p) attenuated the enhanced levels. However, PAE alone and ESC alone group did not show any alterations in the total calcium level. Abbreviations: NC, normal control; DC, diabetic control; D, diabetic; PAE, Phyllanthus amarus extract; ESC, esculetin; STZ, streptozotocin. Protein Content Note: The signiﬁcant differences in between the groups were compared using one-way ANOVA followed by t-test. Data presented as Mean ± SEM The total protein content in control and diabetic rats was (n = 6). represented (Figure 4). The protein level of diabetic rats P < 0.001 against normal group and *p < 0.05, **p < 0.01 against diabetic was lower than the control rats (24.44%, 28.57% and group. 32.6% lower on day 7, 14 and 21, respectively, p < 0.001). Treatment with PAE (–7.35%, –16.92% and –30.64% lower Figure 5. Effect of Phyllanthus amarus Extract and Esculetin on + + on day 7, 14 and 21, respectively, p < 0.05) and esculetin Na -K ATPase Activity (µm of Pi Liberated/hr/g) in Sciatic Nerve (–11.76%, –23.07% and –32.25% lower on day 7, 14 and Tissue of STZ-Induced Diabetic Rats 21, respectively, p < 0.01) significantly attenuated the mentioned effect. Figure 3. Effect of Phyllanthus amarus Extract and Esculetin on Ni- tric Oxide (NO) Level (µg/mg Protein) Nitrite 0.400 NC DC 0.350 # D+PAE D+ESC 0.300 ** PAE 0.250 ESC ** 0.200 ** 0.150 0.100 0.050 0.000 Abbreviations: NC, normal control; DC, diabetic control; D, diabetic; PAE, day 7 day 14 day 21 Phyllanthus amarus extract; ESC, esculetin; STZ, streptozotocin. Experimental days Note: The signiﬁcant differences in between the groups were compared using one-way ANOVA followed by t-test. Data presented as Mean ± SEM (n = 6). Abbreviations: NC, normal control; DC, diabetic control; D, diabetic; PAE, P < 0.001 against normal group and *p, **p < 0.05 against diabetic group. Phyllanthus amarus extract; ESC, esculetin; STZ, streptozotocin. Note: The signiﬁcant differences in between the groups were compared + + using one-way ANOVA followed by t-test. Data presented as Mean ± SEM Na -K ATPase Activity (n = 6). + + # Diabetes resulted in significant decrease in Na -K ATPase P < 0.001 against normal group and *p < 0.05, **p < 0.05 against diabetic group (n = 6). activity (44.03%, 50.27% and 57.73% lower on day 7, 14 g/mg of protein Srilatha and Reddy 23 and 21, respectively, p < 0.001), whereas treating the diabetic (–17.48%, –41.82% and –81.67% lower on day 7, 14 and 21, + + group with Phyllanthus amarus (0.48%, –38.8% and –71.82% respectively, p < 0.05) significantly improved the Na -K lower on day 7, 14 and 21, respectively, p < 0.05) and esculetin ATPase activity in the sciatic nerve tissue (Figure 5). Table 3. Effects of Phyllanthus Amarus Extract and Esculetin on Calcium (Ca) Level (ppm/mg of Protein) in Sciatic Nerve Tissue of STZ-In- duced Diabetic Rats % % % % % Change Change Change Change Change from from from from from Groups NC DC Control D+PAE Control D+ESC Control PAE Control ESC Control Calcium 4.45± 36.67± –724.04 19.5±0.76 –338.20 16.83± –278.20 4.38± –8.53 4.42± 0.67 # ** 0.17 3.43 0.60 0.11 0.11 Note: The signiﬁcant differences in between the groups were compared using one-way ANOVA followed by t-test. Data presented as Mean ± SEM (n = 6). P < 0.001 against normal group and *p, **p < 0.005 against diabetic group. Figure 6a. Samples of NCV Recorded From (a) NC, (b) DC, (c) D+PAE, (d) D+ESC, (e) PAE, (f) ESC (n = 6) Abbreviations: NC, normal control; DC, diabetic control; D, diabetic; PAE, Phyllanthus amarus extract; ESC, esculetin; STZ, streptozotocin. Figure 6b. Effect of Phyllanthus amarus Extract and Esculetin on Nerve Conduction Velocity in Sciatic Nerve Tissue of STZ-Induced Dia- betic Rats (m/s) 60.00 50.00 ** 40.00 30.00 20.00 10.00 0.00 NC DC D+PAE D+ESC PAE ESC Experimental Groups Abbreviations: NC, normal control; DC, diabetic control; D, diabetic; PAE, Phyllanthus amarus extract; ESC, esculetin; STZ, streptozotocin. Note: The signiﬁcant differences in between the groups were compared using one-way ANOVA followed by t-test. Data presented as Mean ± SEM (n = 6). *P < 0.001 against normal group and **p < 0.01, ***p < 0.001 against diabetic group.ç Nerve Conduction Velocity (m/s) 24 Annals of Neurosciences 26(3–4) 8q, 8r). The transverse section of sciatic nerve from the normal control group showed a part of nerve fascicle surrounded by connective tissue, perineurium. It contains axons of various sizes surrounded by myelin sheath and separated by an endoneurial connective tissue. Nuclei of Schwann cells, endoneurium and blood vessels were seen. However, in the diabetic control group, sciatic nerve showed shrinkage, demyelination, necrosis and degenerated nerve fibres surrounded by thin perineurium. Focal loss of endoneurium was also observed due to destructive reactions of STZ three weeks after diabetes induction (Figures 8d, 8e, 8f). Treatment of diabetic rats with Phyllanthus amarus (Figures 8g, 8h, 8i) and esculetin (Figures 8j, 8k, 8l) were able to preserve the normal appearance of axons, myelin spaces and perineurium. Figure 7. Effect of Phyllanthus amarus Extract and Esculetin on Ace- tylcholine Content in Sciatic Nerve Tissue (mg/gm Weight of Tissue) Figure 8. Histological Assessment of T.S of Sciatic Nerve. (a, b, c) of STZ-Induced Diabetic Rats Normal Control (NC); (d, e, f) Diabetic Control (DC); (g, h, i) PAE Received Diabetic Group (D+PAE); (j, k, l) ESC Received Diabetic Abbreviations: NC, normal control; DC, diabetic control; D, diabetic; PAE, Group; (m, n, o) PAE Received Control; (p, q, r) ESC Received Control Phyllanthus amarus extract; ESC, esculetin; STZ, streptozotocin. Note: The signiﬁcant differences in between the groups were compared using one-way ANOVA followed by t-test. Data presented as Mean ± SEM (n = 6). P < 0.005 against normal group and *p < 0.05, **p < 0.05 against diabetic group. Nerve Conduction Velocity When compared with the control group, diabetic group showed decreased (44.44%, p < 0.001) mean nerve conduction velocity (MNCV). There was a significant increase of MNCV in diabetic groups treated with Phyllanthus amarus plant extract (–32.72%, p < 0.01) and esculetin (–41.82%, p < 0.001) in contrast to the diabetic group. These results show that PAE and esculetin have a protective role by improving the MNCV in diabetic neuropathy rats and also show the efficacy of doses at 400 mg/kg b.w/ml/day p.o and 45 mg/kg b.w/ml/day i.p, respectively (Figures 6a and 6b). Acetylcholine Content The acetylcholine content was decreased significantly in sciatic nerve of diabetic rats when compared with the control rats (31.42%, 40.54% and 47.37% lower on day 7, 14 and 21, respectively, p < 0.005). PAE (20.83%, 40.9% and 60% lower on day 7, 14 and 21, respectively, p < 0.05) and ESC (29.17%, 45.45% and 65.00% lower on day 7, 14 and 21, respectively, p < 0.05) treated diabetic rats have significantly attenuated the mentioned effect (Figure 7). Light Microscopy Three weeks after confirmation of diabetes, no pathological Note: p, perineurium; e, endoneurium; bv, blood vessel; black arrow: Schwann changes were observed in control group (Figures 8a, 8b, 8c) rats cell nuclei; curved arrow: axon; green arrow: myelin sheath; haematoxylen received with PAE (Figures 8m, 8n, 8o) and ESC (Figures 8p, and eosin staining (10X, 40X, 40X). Srilatha and Reddy 25 Figure 9. TEM studies for all groups (a, b) normal control (NC); (i, concentric light and dark circles and axonal structures. Sciatic j) PAE received control; (k, l) ESC received control groups showing nerve tissue of diabetic rats showed disorganized myelin normal myelinated (Mn), unmyelinated (Un) nerve ﬁbres, axoplasm sheath. It was enlarged and distorted near axonal and stromal (A) containing microtubules and mitochondria (M) wrapped in a sides (Figures 9c, 9d). Fragmentation of lamellae, and thick myelinated sheath with compact lamellar structure and sur- demyelination and separation of myelin sheath were clearly rounded by Schwann cells. (c, d) Diabetic control (DC) group show- observable. Schwann cells were showing the uneven nuclei, ing shrinkage of cells, myelin breakdown, discontinuous and disor- vacuolar mitochondria, expanded endoplasmic reticulum and ganised myelin sheath (curved arrow), loss of axoplasm, focal lysis disrupted basement membrane. At a dose of 400mg/kg b.w/ (red arrow) of myelin sheath, irregular nuclei in the blood vessels ml/day, p.o of Phyllanthus amarus (Figures 9e, 9f) and 45mg/ (yellow arrow), separation of myelin lamellae, loss of compact lamel- lar structure (double arrow), compressed and distorted axoplasm kg b.w/ml/day, i.p of esculetin (Figures 9g, 9h) alleviated the (A), swollen and destroyed mitochondria (M). (e, f) PAE treated di- vacuolar deficits, and discrete myelin structure and inhibited abetic group (D+PAE); (g, h) ESC treated diabetic (D+ESC) groups the separation of lamellar structures. Increase of Schwann showing many axons with normal appearance of myelin sheath and cell number, myelin and axon density was observed. endoneurium in-between, some focal lysis, focal appearance of re- dundant myelin (blue arrow), separation of myelin sheath from axon (double side arrow), vacuolation of the cytoplasm (V), presence of Discussion some microtubules and mitochondria (M). Scale: 2 µm. The protective effects of hydro-ethanolic PAE plant (400 mg/kg b.w/ml/day p.o) and esculetin (45 mg/kg b.w/ml/day i.p) on diabetic neuropathy in STZ -induced diabetic male wistar rats were investigated in the current study through behavioural, biochemical, electrophysiological and sciatic nerve histopathological studies. Evidence indicates that increased glucose levels in the body has toxic effects on peripheral nerves due to increased glucose auto-oxidation, which leads to increase in the synthesis of reactive species. STZ causes elevation of glucose levels in the blood by selectively destructing the cells of islets of pancreas. In our previous study, rats with STZ-induced diabetes had significantly elevated the blood glucose levels and water intake levels, and decreased body weights compared with the control rats. Behavioural alterations were observed in diabetic rats as soon as diabetes started. Diabetic rats spent less time on rotarod and took more time to reach the goal on maze, compared to the control rats due to synergistic association between metabolic changes related to diabetes and impaired conditions within the peripheral nervous system. These findings are in line with the earlier report. The diabetic rats treated with PAE and ESC showed that improved behaviour may be due to consolidated action of learning and eventual memory formation, and coordination between sensory and motor neurons. The organized activity between the complex sensory and motor neurons was deliberated and stimulated by synapses formation, it’s strength, communication and by the release of neurotransmitters like acetylcholine. It is evident from the present investigation using albino rats that STZ administration (60 mg/kg body weight) causes a diabetogenic response significantly. From the results given Transmission Electron Microscopy in Table 1, diabetic rats exhibit increased and almost doubled The electronic microscopic structures of the sciatic nerve HbA1c level due to the interaction of excess glucose with tissue of normal rats (Figures 9a, 9b) received only PAE haemoglobin to form glycosylated haemoglobin over time (Figures 9i, 9j) and esculetin (Figures 9k, 9l) displayed in diabetes milletus. A significant elevation in glycosylated normal compact structure of nerve fibre with uniform and haemoglobin noticed in diabetic rats was normalized to thick myelin sheath. Lamellae were also arranged with near normal with the administration of ethanolic PAE and 26 Annals of Neurosciences 26(3–4) esculetin after three weeks. Presence of flavonoids, lignans, and humans. Increase in polyol pathway, AR and sorbitol alkaloids, galloatinoids, saponins, terpinoids, flavonoids and dehydrogenase catalysis will convert the extra glucose into glycosides in the aerial parts of Phyllanthus amarus were sorbitol and fructose. As sorbitol is unable to cross the cell maybe responsible to reduce the hyperglycemia levels in membrane, it leads to accumulation, causing hyperosmolarity STZ-induced diabetic rats. Esculetin, was able to maintain as well as concomitant release of aurine, myoinositol and the normal blood glucose level indicates its anti-diabetic adenosine. As a result, ATP synthesis is inhibited resulting + + activity role. HbA1c level was decreased significantly in lowered Na -K ATPase, PKC, weakened axonal transport when treated with P. amarus and esculetin, maintaining the and structural loss of nerves. Phyllanthin, a component of glycosylated haemoglobin in their normal range, and were Phyllanthus amarus, has affinity towards AR enzyme and quite comparable. This proves that the role of Phyllanthus is able to decrease the oxidative stress by enhancing the amarus and esculetin in controlling the blood glucose is synthesis of NADPH. From the results obtained previously, possibly mediated via improvement in insulin secretion from Phyllanthus amarus and esculetin were able to decrease the the existing β-cells of pancreas through increased expression AR activity in diabetic rats in vivo. Further they can restore + + of insulin receptors and an increased flux of glucose into the the nerve conduction velocity by improving Na -K ATPase glycolytic pathway by avoiding the polyol pathway. activity. As a result, the cholinergic activity was improved MPO is an inflammatory marker, a specific reductase for by increasing the synthesis of acetylcholine. Thus, PAE and neutrophils and is expressed abundantly in polymorphonuclear ESC were playing an important role in the deactivation of neutrophils (PMN) in their azurophilic granules. So, polyol pathway by enhancing neurotrophic support in the estimation of MPO expression in the sciatic nerve tissue amelioration of acute diabetic neuropathy. can therefore emulate the degree of extravasation of PMN The histological observations in transverse section of from their microvasculature. The increased MPO levels in sciatic nerve and electron microscopic studies of diabetic diabetic rats have shown the expanse of PMN extravasation as rats have shown shrinkage of cells, demyelination, necrosis well as erratic expansion of neutrophils submitting that there and degenerated nerve fibres surrounded by thin perineurium was an in vivo inflammatory reaction caused due to diabetes. and focal loss of endoneurium, disorganized myelin sheath, The diabetic rats treated with esculetin and PAE rescued the lamellae, uneven Schwann cells with nuclei, vacuolar MPO levels thereby lowering the degree of invasion of PMNs mitochondria, expanded endoplasmic reticulum and disrupted from microvasculature tissue thereby reducing the viscosity basement membrane and these results were parallel with the of blood. previous reports. After confirmation of diabetes, treatment The rise in the total nitrite and calcium levels, and of diabetic rats for three weeks with Phyllanthus amarus and decrease in protein content were observed in the diabetic esculetin were able to retain the normal appearance of axons, group in contrast to the control rats. The increased Ca myelin spaces and perineurium, normal compact structure levels will get accumulated in nerve tissue and activate of nerve fibre with uniform and thick myelin sheath and the secondary messengers. Further, they activate the lamellae, recovery of Schwann cell number and increase of calcium dependent kinases and phosphatases which can myelin, axon density were observed. No pathological changes change the homeostasis in the nervous system leading to were observed in the control group and control rats received unstability of cytoskeletal proteins of axons which leads to with PAE alone and ESC alone. Sciatic nerve from the normal 51,52 their degeneration. Various studies have displayed that control group showed a compact structure with perineurium, calcium, free radical induced oxidative, nitrosative stress and axons surrounded by myelin sheath and separated by an inflammatory mediators play a key role in the progression of endoneurial connective tissue. many neurodegenerative diseases such as Parkinson’s and Esculetin has shown effective activity against diabetes- Alzheimer’s diseases, neuropathic pain, amyotrophic lateral induced adverse effects in comparison with the Phyllanthus sclerosis, pathophysiology of chronic diabetes and are well amarus extract. The results indicated the activity of pure reported in chronic constriction injury and axotomy of sciatic compounds against a crude compound even though both nerve. Reduced levels of Ca, NO and increased levels of PAE and ESC had almost similar effect against STZ-induced protein content were observed in PAE and ESC treated diabetes (Figure 10). Coumarins and coumarin-derived diabetic rats may be attributed to their anti-oxidative, anti- compounds possess an extensive pharmaceutical and nitrosative and anti-inflammatory roles. biological action and have prominent significance in the Besides biochemical and behavioural effects, PAE treatment of various diseases and were clinically very and esculetin have significantly restored the sciatic nerve significant. They exhibit notable pharmacokinetic activity conduction velocity and were able to recover the nerve due to their rapid absorption and metabolism in the body. In structure in STZ-induced neuropathic rats. Slowing down the present study, esculetin (6,7-dihydroxy coumarin) proved of nerve conduction velocity is an early and usual sign in a significant effect against diabetic neuropathy by its anti- neuronal defects and dysfunction as observed in diabetic rats oxidant, anti-diabetic and anti-inflammatory activity. Srilatha and Reddy 27 Figure 10. Possible Mechanism Showed by PAE and ESC in Preventing the Progression of DPN Ethanolic extract of Phyllanthus amarus Esculetin 21 days Anti-diabetic, STZ 60mg/kg BW Antioxidant & Anti treatment inflammatory Diabetic Free radicals Free radicals Na+/K+-ATPase HbA1c Na+/K+-ATPase HbA1c NCV MPO NCV MPO Ach Ca Ach Ca Protein NO Protein NO Motor coordination Motor coordination Maze learning ability Maze learning ability Prevention of initiation of adverse Structural & Functional processes of s ciatic nerve tissue impairment of sciatic nerve tissue The hydro-ethanolic PAE was found to show a sensitive Acknowledgement and preventive response on diabetic-induced effects at 400mg/ Authors would like to thank UGC-DSA-I SAP-II. Lr. No. F.5- kg b.w concentration. Reduction in the glucose level, which 26/2015/DSA-1 (SAP-II) programme for the partial financial ultimately regulates polyol pathway so that the usual function assistance. of the sciatic nerve is attained with proper nerve conduction velocity, was maybe the mechanism involved. The biological Author Contributions activity of Phyllanthus amarus using as herbal medicine Srilatha Kota designed and performed the work and also prepared appears to be obtained from secondary metabolites having this article. Pratap Reddy Karnati designed, checked the progression therapeutic activity such as coumarins, lignans, flavonoids, of work, discussed the results and conclusion, and participated in the triterpenoids, vitamin C and many alkaloids. The obtained preparation of this article. results have demonstrated the importance of these metabolites significantly as a therapeutic approach. Ethical Statement Procedures of the present study were duly approved by Animal Conclusion Ethics Committee, Osmania University, Hyderabad, Telangana, India. On the basis of the results obtained, it can be suggested that Phyllanthus amarus and esculetin produced a protective effect Declaration of Conflicting Interests on STZ -induced DPN by the prevention of initiation of adverse The authors declared no potential conflicts of interest with processes of sciatic nerve, and the possible recovery of internal respect to the research, authorship, and/or publication of this and external structures could be accredited to their multifarious article. effects such as anti-diabetic, anti-oxidative, anti-inflammatory and neuroprotective roles manifested in terms of mitigation of behavioural, biochemical and histopathological changes. PAE and Funding ESC have shown almost similar effects. 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Annals of Neurosciences – SAGE
Published: Jul 1, 2019