Mitigation of lens opacification by a functional food in a diabetic rodent model.

The current study was designed to test a functional food (FF) mixture containing aldose reductase inhibitors and antiglycation bioactive compounds for suppressing the onset and progression of cataracts in a diabetic rat model. Two-month-old Sprague Dawley rats were grouped as control (C), diabetes untreated (D), and diabetic rats treated with FF at two doses (FF1 = 1.35 g and FF2 = 6.25 g/100g of diet). Diabetes was induced by a single injection of streptozotocin. The FF is a mixture of amla, turmeric, black pepper, cinnamon, ginger, and fenugreek added to the rodent diet. The status of cataracts was monitored weekly by a slit lamp examination for 20 weeks, after which animals were sacrificed to collect eye lenses. Feeding FF1 and FF2 to diabetic rats yielded a significant anti-hyperglycaemic effect and marginally prevented body weight loss. FF delayed cataract progression, and FF2 showed better efficacy than FF1. FF prevented the loss of lens crystallins and their insolubilization in diabetic rats. The antioxidant potential of FF was evident with the lowered protein carbonyls, lipid peroxidation, and prevention of altered antioxidant enzyme activities induced by diabetes. These studies demonstrate the efficacy of plant-derived dietary supplements against the onset and progression of cataracts in a well-established rat model of diabetic eye disease.

Dietary zinc inadequacy affects neurotrophic factors and proteostasis in the rat brain.

Zinc (Zn) deficiency has many adverse effects, including growth retardation, loss of appetite, vascular diseases, cognitive and memory impairment, and neurodegenerative diseases. In the current study, we investigated the hypothesis that dietary Zn inadequacy affects neurotrophic factors and proteostasis in the brain. Three-week-old Wistar/Kyoto male rats were fed either a Zn-deficient diet (D; < 1 mg Zn/kg diet; n = 18) or pair-fed with the control diet (C; 48 mg Zn/kg diet; n = 9) for 4 weeks. Subsequently, the rats in the D group were subdivided into two groups (n = 9), in which one group continued to receive a Zn-deficient diet, whereas the other received a Zn-supplemented diet (R; 48 mg Zn/kg diet) for 3 more weeks, after which the rats were sacrificed to collect their brain tissue. Markers of endoplasmic reticulum stress, ubiquitin-proteasome system, autophagy, and apoptosis, along with neurotrophic factors, were investigated by immunoblotting. Proteasomal activity was analyzed by the spectrofluorometric method. The results showed an altered ubiquitin-proteasome system and autophagy components and increased gliosis, endoplasmic reticulum stress, and apoptosis markers in Zn-deficient rats compared with the control group. Zinc repletion for 3 weeks could partially restore these alterations, indicating a necessity for an extended duration of Zn supplementation. In conclusion, a decline in Zn concentrations below a critical threshold may trigger multiple pathways, leading to brain-cell apoptosis.

Anti-inflammatory potential of turmeric, amla, and black pepper mixture against sepsis-induced acute lung injury in rats.

Acute lung injury (ALI), is a severe inflammatory lung disease. We tested the prophylactic effect of a functional food mix comprising three anti-inflammatory plant products: turmeric, amla, and black pepper (TAB) against lipopolysaccharide (LPS)-induced ALI in rats. Two-month-old male Wistar rats were randomly divided into three groups: control (C), LPS (5 mg/kg), and LPS with TAB (TAB). After 6 h of LPS injection, the rats were sacrificed by cervical decapitation to collect the lung tissue. Results showed that TAB partially ameliorated LPS-induced increase in circulating inflammatory cytokines (TNFα and IL6) and significantly prevented lung histopathological changes. TAB also suppressed LPS-activated ER stress markers (GRP78, pIRE1, and CHOP) and apoptotic markers (caspase-3 and - 12) in the lung. The anti-inflammatory effects of the TAB support its potential use as an adjuvant to mitigate ALI. Importantly, TAB's ingredients have been used for centuries as part of the diet with limited or no toxic effects.

Gut mycobiome dysbiosis in rats showing retinal changes indicative of diabetic retinopathy.

The current study compared the gut mycobiomes of diabetic rats generated by a streptozotocin chemical challenge, diabetic rats with retinal changes and normal control rats over a period of 4 months. Sustained increase in blood sugar levels (>150 mg/dL) confirmed the induction of diabetes. Histology and immunohistochemistry were used to identify changes in the retinal tissues in the diabetic rats indicative of the animals progressing into diabetic retinopathy. Gut mycobiomes generated using faecal DNA, indicated dysbiosis at the genus level in both diabetic (DM) and diabetic rats with retinal changes (DRC) when compared with the control rats. In Tables 3-6 the specific genera that were significantly increased/decreased in DM1 and DM2 and in DRC1 and DRC2 respectively compared to the respective controls CT1-CT4 rats are listed. Further, the mycobiomes of the DM and DRC rats separated into distinct clusters following heat-map analysis of the discriminating genera. In addition, ß-diversity analysis separated the mycobiomes of DM and DRC rats from that of the control rats, but the mycobiomes of diabetic rats and diabetic rats with retinal changes showed an overlap. Based on the inferred functions of the discriminating genera in the mycobiomes, we speculated that increase in pathogenic fungi might contribute to the inflammatory status both in diabetic rats and rats showing retinal changes.

Age-related neuronal damage by advanced glycation end products through altered proteostasis.

Aging is a main risk factor for many diseases including neurodegenerative disorders. Numerous theories and mechanisms including accumulation of advanced glycation end products (AGEs) have been put forward in explaining brain aging. However, a focused study on the status of AGEs in the brain during progressive aging in connection with interrelated cellular processes like ubiquitin-proteasome system (UPS), unfolded protein response, autophagy-lysosome system and apoptosis is lacking. In this study, we investigated the levels of AGEs in the brain of 5-, 10-, 15- and 20-months old WNIN rats. Endoplasmic reticulum (ER) stress response, UPS components, autophagy flux, neurotrophic and presynaptic markers along with cell death markers were analyzed by immunoblotting. The neuronal architecture was analyzed by H&E and Nissl staining. The results demonstrated progressive accumulation of AGEs in the brain during aging. Adaptive ER stress response was observed by 10-months while maladaptive ER stress response was seen at 15- and 20-months of age along with impaired UPS and autophagy, and perturbations in neuronal growth factors. All these disturbances intensify with age to further exaggerate cell death mechanisms. There was a shrinkage of the cell size with aging and Congo-red staining revealed ß-amyloid accumulation in higher ages. Together these results suggest that progressive accumulation of AGEs with aging in the brain may lead to neuronal damage by affecting ER homeostasis, UPS, autophagic flux, and neuronal growth factors.

Dysbiosis in the Gut Microbiome in Streptozotocin-Induced Diabetes Rats and Follow-Up During Retinal Changes.

Purpose: To analyze the gut bacterial microbiome of streptozotocin-induced diabetic rats and rats with retinal changes. Methods: Induction of diabetes was confirmed by an increase in blood sugar (>150 mg/dL), and the progression of diabetes with retinal changes was assessed by histology and immunohistochemistry of retinal sections. Microbiomes were generated using fecal DNA, and the V3-V4 amplicons were sequenced and analyzed by QIIME and R. Results: Dysbiosis in the gut microbiome of diabetic rats and diabetic rats with retinal changes was observed at the phylum and genus levels compared with the control rats. Heat-map analysis based on the differentially abundant genera indicated that the microbiomes of controls and diabetic rats separated into two distinct clusters. The majority of the microbiomes in diabetic rats with retinal changes also formed a distinct cluster from the control rats. ß-diversity analysis separated the microbiome of control rats from the microbiome of diabetic rats and diabetic rats with retinal changes, but the microbiomes of diabetic rats and diabetic rats with retinal changes showed an overlap. Functional analysis indicated that the enhanced inflammation in diabetic rats showing retinal changes could be ascribed to a decrease in anti-inflammatory bacteria and an increase in pathogenic and proinflammatory bacteria. Conclusions: This study showed that the gut bacterial microbiome in diabetic rats with retinal changes was different compared with control rats. The results could help develop novel therapeutics for diabetics and diabetic individuals with retinal changes.

Effect of vitamin B12 supplementation on retinal lesions in diabetic rats.

Purpose: Diabetic retinopathy (DR) is the most common complication of diabetes involving microvasculature and neuronal alterations in the retina. Previously, we reported that vitamin B12 deficiency could be an independent risk factor for DR in humans. However, the effect of vitamin B12 supplementation in experimental DR is unknown. Thus, in this study, we investigated the impact of dietary supplementation of vitamin B12 on retinal changes in diabetic rats. Methods: Diabetes was induced in 2-month-old Sprague-Dawley rats and maintained for 4 months. One group of diabetic rats were fed normal levels of vitamin B12, and one group double the quantity of vitamin B12 (50 µg/kg diet). Vitamin B12 and homocysteine levels in the plasma were analyzed with radioimmunoassay (RIA) and high-performance liquid chromatography (HPLC), respectively. At the end of 4 months of experimentation, the eyeballs were collected. Retinal changes were analyzed with hematoxylin and eosin (H&E) staining, immunoblotting, and immunofluorescence methods. Results: Dietary supplementation of vitamin B12 had no effect on food intake, bodyweight, fasting blood glucose, and plasma homocysteine levels in the diabetic rats. However, vitamin B12 supplementation prevented loss of rhodopsin, and overexpression of VEGF, and completely prevented overexpression of HIF1α, GFAP, and endoplasmic reticulum (ER) stress markers (GRP78, ATF6α, XBP1, CHOP, and caspase 12) in the diabetic rat retina. Further, vitamin B12 ameliorated apoptosis in the retina as shown with terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) and prevented retinal thinning. Conclusions: Vitamin B12 supplementation of diabetic rats appeared to be beneficial by circumventing retinal hypoxia, VEGF overexpression, and ER stress-mediated cell death in the retina. The present study adds another potential therapeutic strategy of vitamin B12 in diabetes.

Spatio-temporal control on the delivery of triamcinolone acetonide using polymeric nanoparticles reduces steroid induced cataract.

Development of topically administered drug delivery systems for the treatment of ocular diseases have majorly focused on enhancing bioavailability of drugs in the ocular tissues. However, control of spatial distribution of topically administered drugs so as to restrict/avoid drug bioavailability at sensitive ocular tissues that are prone to drug induced adverse effects has not been explored. In this study, we aimed to reduce the bioavailability of topically administered corticosteroid, triamcinolone acetonide (TA) in lens via controlled spatial distribution in order to minimize TA induced posterior subcapsular cataract (PSC). For this, a negatively charged polymeric core-shell nanoparticulate drug delivery system composed of polycaprolactone (PCL) core and pluronic® F-68 (PF68) shell was fabricated. For in vivo studies, coumarin-6 (COU) loaded nanoparticles (NPs) were fabricated and studied for their biodistribution after topical administration in mice eyes and compared with free COU biodistribution. The administered COU loaded NPs differentially distributed in mice eyes and showed lower bioavailability in lens compared to free COU. Further, in vivo efficacy of the delivery system for its ability to minimize the rate of PSC progression was evaluated in diabetic rats. The results demonstrated that TA loaded PCL-PF68 NPs decreased PSC progression compared to free TA when administered topically.

4-PBA prevents diabetic muscle atrophy in rats by modulating ER stress response and ubiquitin-proteasome system.

PURPOSE: Skeletal muscle is severely affected in diabetes leading to muscle atrophy. Previously we reported the role of ER stress in muscle atrophy due to hyperglycemia. Hence, in the present study, we investigated the effect of a classical ER stress inhibitor, 4-phenylbutyric acid (PBA), on muscle atrophy in diabetic rats. METHODS: Diabetes was induced in male rats by streptozotocin, and PBA was administered (40 mg/kg/day; intraperitoneal) after two months of diabetes for two more months. Gastrocnemius muscle is collected after four months of experimental period. The cross-sectional area of myocytes was measured on Hematoxylin and Eosin stained muscle sections. Protein levels of ER stress markers, ubiquitin-proteasome system (UPS) components, and apoptosis were analysed by immunoblot. Proteasomal activity and apoptotic cells were measured. RESULTS: ER stress markers (GRP78, ATF6, ATF4 and CHOP) that are elevated in diabetes are decreased with PBA treatment. PBA also averted diabetes-induced alterations in UPS (higher levels of E1, atrogin-1, UCHL1 and UCHL5, accumulation of ubiquitinated proteins and increased proteasomal activity). Apoptosis mediators-p53, BAX, and cleaved caspase-3 protein levels, and TUNEL positive cells were decreased in PBA treated diabetic rats. PBA notably improved the muscle-cross sectional area. CONCLUSIONS: Results highlighted the therapeutic potential of PBA in diabetes muscle wastage.

Ubiquitin-proteasome system and ER stress in the brain of diabetic rats.

The ubiquitin-proteasome system (UPS) has been implicated in the pathogenesis of many neurodegenerative diseases. Endoplasmic reticulum (ER) stress is shown to play a pathological role in the development of diabetes and its complications. Hence, the current study is aimed to investigate the role of UPS and ER stress in the cerebral cortex of diabetic rats and examine the therapeutic effect of 4-phenylbutyric acid (4-PBA), an ER stress inhibitor. Male Sprague-Dawley rats were divided into three groups: control, diabetes, and diabetes plus 4-PBA treatment group. Diabetes was induced by single intraperitoneal streptozotocin injection (37 mg/kg body weight [bw]), and 4-PBA was administered (40 mg/kg bw/d, intraperitoneal) for 2 months, starting from 2 months of diabetes induction. At the end of 4 months, cerebral cortex was collected for analysis. Declined proteasome activity and ubiquitin C-terminal hydrolase (UCH)-L1 expression, increased ubiquitinated proteins, and apoptosis were observed in the diabetic rats. The expression of the ubiquitin-activating enzyme E1, UCHL5, and ER stress markers (ATF6, pPERK, and CHOP) was markedly elevated, whereas the expression of ER-associated protein degradation (ERAD) components was downregulated in the diabetic rats. 4-PBA intervention attenuated ER stress, alterations in UPS, and ERAD components in diabetic rats. Importantly, neuronal apoptosis was lowered in 4-PBA-treated diabetic rats. Our observations demonstrate that altered UPS could be one of the underlying mechanisms of neuronal apoptosis in diabetes and chemical chaperones such as 4-PBA could be potential candidates for preventing these alterations under hyperglycemic conditions.

Procyanidin-B2 enriched fraction of cinnamon acts as a proteasome inhibitor and anti-proliferative agent in human prostate cancer cells.

Altered activity of the proteolytic machine-the 26S proteasome is observed in many disease conditions. Hence, either inhibition or activation of the 26S proteasome is thought to be a novel therapy for treatment of certain diseases such as cancer and neurodegenerative disorders. In this study, we tested the potential of cinnamon and one of its active ingredients, procyanidin-B2 (PCB2), in inhibiting the catalytic activities of the proteasome and suppressing prostate cancer cell growth. Proteasome activities were measured using fluorogenic substrates specific for the different enzymatic activities of the 26S proteasome by flourometry. Cell viability was assessed using the 3-[4, 5-dimethylthiazol-2-yl]-2.5-diphenyl-tetrazolium bromide assay, while apoptosis was examined by Hoechst and propidium iodide staining and caspase-3 activity. Both, the cinnamon extract and its PCB2-enriched F2 fraction inhibited the catalytic activities of the purified proteasome and the proteasome in cancer cells but not in normal cells. Furthermore, cinnamon and its active component decreased cell proliferation of human prostate cancer cells but not normal lung cells, decreased expression of anti-apoptotic and angiogenic markers in prostate cancer cell lysates. These results demonstrate that cinnamon extract and its PCB2-enriched fraction act as proteasome inhibitors and have prospects as anti-cancer agents. © 2018 IUBMB Life, 70(5):445-457, 2018.

Implication of altered ubiquitin-proteasome system and ER stress in the muscle atrophy of diabetic rats.

BACKGROUND: Skeletal muscle is adversely affected in type-1 diabetes, and excessively stimulated ubiquitin-proteasome system (UPS) was found to be a leading cause of muscle wasting or atrophy. The role of endoplasmic reticulum (ER) stress in muscle atrophy of type-1 diabetes is not known. Hence, we investigated the role of UPS and ER stress in the muscle atrophy of chronic diabetes rat model. METHODS: Diabetes was induced with streptozotocin (STZ) in male Sprague-Dawley rats and were sacrificed 2- and 4-months thereafter to collect gastrocnemius muscle. In another experiment, 2-months post-STZ-injection diabetic rats were treated with MG132, a proteasome inhibitor, for the next 2-months and gastrocnemius muscle was collected. RESULTS: The muscle fiber cross-sectional area was diminished in diabetic rats. The expression of UPS components: E1, MURF1, TRIM72, UCHL1, UCHL5, ubiquitinated proteins, and proteasome activity were elevated in the diabetic rats indicating activated UPS. Altered expression of ER-associated degradation (ERAD) components and increased ER stress markers were detected in 4-months diabetic rats. Proteasome inhibition by MG132 alleviated alterations in the UPS and ER stress in diabetic rat muscle. CONCLUSION: Increased UPS activity and ER stress were implicated in the muscle atrophy of diabetic rats and proteasome inhibition exhibited beneficiary outcome.

Amelioration of neuronal cell death in a spontaneous obese rat model by dietary restriction through modulation of ubiquitin proteasome system.

Dietary restriction (DR) has been shown to increase longevity, delay onset of aging, reduce DNA damage and oxidative stress and prevent age-related decline of neuronal activity. We previously reported the role of altered ubiquitin proteasome system (UPS) in the neuronal cell death in a spontaneous obese rat model (WNIN/Ob rat). In this study, we investigated the effect of DR on obesity-induced neuronal cell death in a rat model. Two groups of 40-day-old WNIN/Ob rats were either fed ad libitum (Ob) or pair-fed with lean. The lean phenotype of WNIN/Ob rats served as ad libitum control. These animals were maintained for 6.5months on their respective diet regime. At the end of the study, cerebral cortex was collected and markers of UPS, endoplasmic reticulum (ER) stress and autophagy were analyzed by quantitative real-time polymerase chain reaction, immunoblotting and immunohistochemistry. Chymotrypsin-like activity of proteasome was assayed by the fluorimetric method. Apoptotic cells were analyzed by TUNEL assay. DR improved metabolic abnormalities in obese rats. Alterations in UPS (up-regulation of UCHL1, down-regulation of UCHL5, declined proteasomal activity), increased ER stress, declined autophagy and increased expression of α-synuclein, p53 and BAX were observed in obese rats and DR alleviated these changes in obese rats. Further, DR decreased TUNEL-positive cells. In conclusion, DR in obese rats could not only restore the metabolic abnormalities but also preserved neuronal health in the cerebral cortex by preventing alterations in the UPS.

Altered ubiquitin-proteasome system leads to neuronal cell death in a spontaneous obese rat model.

BACKGROUND: Obesity is associated with various progressive age-related diseases, including neurological disorders. However, underlying molecular basis for increased risk of neurodegeneration in obesity is unknown. A suitable animal model would immensely help in understanding the obesity-linked neurological problems. METHODS: A spontaneously developed obese rat (WNIN/Ob) which is highly vulnerable for a variety of degenerative diseases was isolated from the existing WNIN stock rats. Ultrastructure of neurons in the cerebral cortex of 12-month old obese rats was evaluated by transmission electron microscopy. qRT-PCR and immunoblotting of ubiquitin C-terminal hydrolases (UCHs), ubiquitin, proteasomal sub-units, markers of ER stress and apoptosis were performed in the cerebral cortex. Proteasome activity was assayed by fluorometric method. Immunohistochemistry was performed for mediators of apoptosis, which was further confirmed by TUNEL assay. These investigations were also carried in high-fat diet-induced obese rat model. RESULTS: Neurons in the cerebral cortex of 12-month obese rats showed swollen mitochondria, disrupted ER and degenerating axons, nucleus and finally neurons. Results showed altered UPS, existence of ER stress, up-regulation of apoptotic markers and apoptosis in the cerebral cortex of obese rats. It appears that UCHL-1 mediated apoptosis through stabilizing p53 might play a role in neuronal cell death in obese rat. Similar changes were observed in the brain of diet-induced obese WNIN rats. CONCLUSION: Altered UPS could be one of the underlying mechanisms for the neuronal cell death in obese conditions. GENERAL SIGNIFICANCE: This is the first report to highlight the role of altered UPS in neurodegeneration due to obesity.

Beneficiary effect of Tinospora cordifolia against high-fructose diet induced abnormalities in carbohydrate and lipid metabolism in Wistar rats.

High intake of dietary fructose has been shown to exert a number of adverse metabolic eff ects in humans and experimental animals. The present study was designed to investigate the eff ect of the aqueous extract of Tinospora cordifolia stem (TCAE) on the adverse eff ects of fructose loading toward carbohydrate and lipid metabolism in rats. Adult male Wistar rats of body weight around 200 g were divided into four groups, two of which were fed with starch diet and the other two with high fructose (66 %) diet. Plant extract of TC (400 mg/kg/day) was administered orally to each group of the starch fed rats and the highfructose fed rats. At the end of 60 days of experimental period, biochemical parameters related to carbohydrate and lipid metabolism were assayed. Hyperglycemia, hyperinsulinemia, hypertriglyceridemia, insulin resistance, and elevated levels of hepatic total lipids, cholesterol, triglycerides, and free fatty acids (p < 0.05) observed in fructose-fed rats were completely prevented with TCAE treatment. Alterations in the activities of enzymes of glucose metabolism (hexokinase, phosphofructokinase, pyruvate kinase, glucose-6-phosphatase, fructose-1,6-bisphosphatase, and glucose-6-phosphate dehydrogenase) and lipid metabolism (fatty acid synthetase, lipoprotein lipase, and malic enzyme) as observed in the high fructose-fed rats were prevented with TCAE administration. In conclusion, our fi ndings indicate improvement of glucose and lipid metabolism in high-fructose fed rats by treatment with Tinospora cordifolia, and suggest that the plant can be used as an adjuvant for the prevention and/or management of insulin resistance and disorders related to it.

Antioxidant potential of aqueous extract of Phyllanthus amarus in rats.

OBJECTIVE: Increased levels of oxidative stress may be implicated in the etiology of many pathological conditions. Protective antioxidant action imparted by many plant extracts and plant products make them promising therapeutic drugs for free radical induced pathologies. In this study we assessed the antioxidant potential of Phyllanthus amarus (Euphorbiaceae). MATERIALS AND METHODS: EXPERIMENTAL RATS WERE DIVIDED INTO TWO GROUPS: Control and Phyllanthus amarus (P. amarus) treated. Treated rats received P. amarus aqueous extract (PAAEt) at a dose of 200 mg/kg body wt/day for 8 weeks. After the treatment period of 8 weeks lipid peroxidation (LPO), vitamin C, uric acid and reduced glutathione (GSH) were estimated in plasma and antioxidant enzymes: Glutathione peroxidase (GPx), catalase (CAT) and superoxide dismutase (SOD) were also assayed. Genotoxicity of PAAEt was assessed by single cell gel electrophoresis (SCGE) of lymphocytes under both in vitro and in vivo conditions. The protective role of PAAEt against hydrogen peroxide (H(2)O(2)), streptozotocin (STZ) and nitric oxide generating system induced lymphocyte DNA damage was also assessed by SCGE. RESULTS: PAAEt treated rats showed a significant decrease in plasma LPO and a significant increase in plasma vitamin C, uric acid, GSH levels and GPx, CAT and SOD activities. SCGE experiment reveals that PAAEt was devoid of genotoxicity and had a significant protective effect against H(2)O(2), STZ and nitric oxide (NO) induced lymphocyte DNA damage. CONCLUSION: The results suggest the non-toxic nature of PAAEt and consumption of PAAEt can be linked to improved antioxidant status and reduction in the risk of oxidative stress.