Impaired renal ischemia reperfusion recovery after bilateral renal artery ligation in rats treated with adenine: role of renal mitochondria.

Vascular calcification (VC) and ischemia reperfusion (IR) injury is characterised to have mitochondrial dysfunction. However, the impact of dysfunctional mitochondria associated with vascular calcified rat kidney challenged to IR is not explored and is addressed in the present study. Male Wistar rats were treated with adenine for 20 days to induce chronic kidney dysfunction and VC. After 63 days, renal IR protocol was performed with subsequent recovery for 24 h and 7 days. Various mitochondrial parameters and biochemical assays were performed to assess kidney function, IR injury and its recovery. Adenine-induced rats with VC, decreased creatinine clearance (CrCl), and severe tissue injury demonstrated an increase in renal tissue damage and decreased CrCl after 24 h of IR (CrCl in ml: IR-0.220.02, VC-IR-0.050.01). Incidentally, the 24 h IR pathology in kidney was similar in both VC-IR and normal rat IR. But, the magnitude of dysfunction was higher with VC-IR due to pre-existing basal tissue alterations. We found severed deterioration in mitochondrial quantity and quality supported by low bioenergetic function in both VC basal tissue and IR challenged sample. However, post 7 days of IR, unlike normal rat IR, VC rat IR did not improve CrCl and corresponding mitochondrial damage in terms of quantity and its function were observed. Based on the above findings, we conclude that IR in VC rat adversely affect the post-surgical recovery, mainly due to the ineffective renal mitochondrial functional restoration from the surgery.

Evaluating the effects of carbon monoxide releasing molecule-2 against myocardial ischemia-reperfusion injury in ovariectomized female rats.

PURPOSE: Cardioprotective effect of carbon monoxide, a gasotransmitter against myocardial ischemia-reperfusion injury (I/R), is well established in preclinical studies with male rats. However, its ischemic tolerance in post-menopausal animals has not been examined due to functional perturbations at the cellular level. METHODS: The protective role of carbon monoxide releasing molecule-2 (CORM-2) on myocardial I/R was studied in female Wistar rats using the Langendorff apparatus. The animals were randomly divided into normal and ovariectomized (Ovx) female rats and were maintained 2 months post-surgery. Each group was further divided into 4 subgroups (n = 6/subgroup): normal, I/R, CORM-2-control (20 µmol/L), and CORM-2-I/R. The cardiac injury was estimated via myocardial infarct size, lactate dehydrogenase, and creatine kinase levels in coronary effluent and cardiac hemodynamic indices. Mitochondrial functional activity was assessed by measuring mitochondrial electron transport chain enzyme activities, swelling behavior, mitochondrial membrane potential, and oxidative stress. RESULTS: Hemodynamic indices were significantly lower in ovariectomized rat hearts than in normal rat hearts. Sixty minutes of reperfusion of ischemic heart exhibited deteriorated cardiac physiological recovery in both ovariectomized and normal groups, where prominent decline was observed in ovariectomized rat. However, preconditioning the isolated heart with CORM-2 improved hemodynamics parameters significantly in both ovariectomized and normal rat hearts challenged with I/R, but with a limited degree of protection in ovariectomized rat hearts. The protective effect of CORM-2 was further confirmed via a reduction in cardiac injury, preservation of mitochondrial enzymes, and reduction in oxidative stress in all groups. CONCLUSION: CORM-2 administration significantly attenuated myocardial I/R injury in ovariectomized rat hearts by attenuating I/R-associated mitochondrial perturbations and reducing oxidative stress.

Vasorelaxant property of Plectranthus vettiveroides root essential oil and its possible mechanism.

ETHNOPHARMACOLOGICAL RELEVANCE: Plectranthus vettiveroides (Jacob) N.P. Singh & B.D. Sharma is a traditional medicinal plant used in Siddha System of Medicine and its aromatic root is used to reduce the elevated blood pressure. AIM: The aim of the present study was to study vasorelaxant property of the root essential oil nanoemulsion (EON) of P. vettiveroides. METHODS: The EON was formulated to enhance the solubility and bioavailability and characterized. The preliminary screening was performed by treating the EON with aortic rings pre-contracted with phenylephrine (1 µM) and potassium chloride (80 mM). The role of K⁺ channels in EON induced vasorelaxation was investigated by pre-incubating the aortic rings with different K⁺ channel inhibitors namely, glibenclamide (a non-specific ATP sensitive K⁺ channel blocker, 10 µM), TEA (a Ca2⁺ activated non-selective K⁺ channel blocker, 10-2 M), 4-AP (a voltage-activated K⁺ channel blocker, 10-3 M) and barium chloride (inward rectifier K⁺ channel blocker, 1 mM). The involvement of extracellular Ca2+ was performed by adding cumulative dose of extracellular calcium in the presence and absence of EON and the concentration-response curve (CRC) obtained is compared. Similarly, the role of nitric oxide synthase, muscarinic and prostacyclin receptors on EON induced vasorelaxation were evaluated by pre-incubating the aortic rings with their inhibitors and the CRC obtained in the presence and absence of inhibitor were compared. RESULTS: The GC-MS and GC-FID analyses of the root essential oil revealed the presence of 62 volatile compounds. The EON exhibited significant vasorelaxant effect through nitric oxide-mediated pathway, G-protein coupled muscarinic (M3) receptor pathway, involvement of K+ channels (KATP, KIR, KCa), and blocking of the calcium influx by receptor-operated calcium channel. CONCLUSION: It is concluded that the root essential oil of P. vettiveroides is possessing marked vasorelaxant property. The multiple mechanisms of action of the essential oil of P. vettiveroides make it a potential source of antihypertensive drug.

Root essential oil of Chrysopogon zizanioides relaxes rat isolated thoracic aorta - an ex vivo approach.

Root of Chrysopogon zizanioides (L.) Roberty has been used in Siddha system of medicine to treat hypertension. The present study was therefore to investigate the vasorelaxation effect of root essential oil of C. zizanioides using rat isolated thoracic aortic rings. Chemical characterization of root essential oil was carried out using Gas Chromatography-Flame Ionization Detector (GC-FID) and Gas Chromatography-Mass Spectrometry (GC-MS). Essential oil nanoemulsion (EONE) was prepared and characterized. Vasorelaxant effect of EONE in endothelium-intact aortic rings precontracted with phenylephrine (PE) (1 µM) or KCl (80 mM) was investigated. Role of Ca2+, nitric oxide and K+ channels in precontracted aortic rings were investigated to elucidate the mechanism of action of the essential oil. Further, the role of muscarinic and prostacyclin receptors in EONE induced relaxation was studied. The EONE significantly induced relaxation (Emax 77.1 ± 4.87%) in PE precontracted aortic rings. The nitric oxide synthase, and cyclooxygenase inhibitors and potassium channel blockers have not significantly inhibited the vasorelaxation induced by EONE. However, EONE induced relaxation in precontracted endothelium-intact aortic rings was significantly inhibited by muscarinic receptor and calcium channel. The root essential oil of C. zizanioides possesses vasorelaxant effect through muscarinic pathway as well as acts as calcium channel blocker.

Radiation-induced H3K9 tri-methylation in E-cadherin promoter during lung EMT: in vitro and in vivo approaches using vanillin.

Radiotherapy is an important treatment regime for lung cancer, worldwide. However, radiation-induced pneumonitis and fibrosis are the treatment-limiting toxicities among patients who have undergone radiotherapy. The epithelial cells via epithelial to mesenchymal transition [EMT] acquires mesenchymal phenotype, which ultimately leads to fibrosis. Many investigations are focussed on understanding the signalling pathways mediating in EMT, however, the role of histone methylation is less understood in radiation-induced lung EMT. In the present study, we analysed the effect of vanillin, an antioxidant, on histone methylation during radiation-induced EMT. The thoracic region of Wistar rats was irradiated with a fractionated dose of X-ray (3 Gy/day) for two weeks (total of 30 Gy). The irradiated animals were sacrificed at the 8th and 16th weeks and tissues were used for analyses. Our data showed that radiation decreased the level of antioxidant enzymes such as SOD, catalase and reduced glutathione that would ultimately enhance oxidative stress in the tissues. Histopathological analysis revealed that radiation increased the infiltration of inflammatory cells to the tissue injury site. Total global histone methylation was increased upon irradiation, which was effectively prevented by vanillin administration. Vanillin enhanced E-cadherin expression and decreased the mesenchymal markers N-cadherin and vimentin in the irradiated lung tissue. The ChIP-qPCR analysis suggested that snail expression in the nucleus might involve in the enrichment of suppressive marker H3K9me3 on the E-cadherin promoter. Finally, we suggested that vanillin administration decreased radiation-induced oxidative stress and EMT expression. Additionally, irradiation increased the H3K9 methylation status with nuclear translocation of snail during lung EMT.

Molecular chemoprevention by morin - A plant flavonoid that targets nuclear factor kappa B in experimental colon cancer.

Colorectal cancer is the third most common cancer worldwide. The development of effective, inexpensive and safe chemopreventive agents would be of great benefit as it involves use of natural products to prevent or suppress the progression of precursor lesions. Morin a flavonoid found in figs (Ficus carica) and other plants is shown to inhibit 1,2-dimethylhydrazine (DMH) induced colon cancer progression in a short term and long term model of colon cancer rats; however, the molecular target for the colon cancer chemoprotective efficacy of morin is yet to be discovered. The present study aims to explore the molecular basis of how morin contributes to the chemoprevention with a focus on NF-κB signaling pathway. The effect of morin on NF-κB signaling in DMH-induced carcinogenic events such as inflammation and apoptosis were analyzed by studying the histopathological analysis using Hematoxylin and Eosin staining (H &E), mRNA expression using q-PCR, protein expression using Immunohistochemistry (IHC) and western blot. Morin supplementation to DMH administered rats down regulated NF-κB pathway and its downstream inflammatory mediators like tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), cyclooxygenase 2 (COX-2) and prostaglandin (PGE-2). Morin supplementation to DMH administered rats alters BAX/BCL2 ratio favoring apoptosis in carcinogen treated rats. Our findings explored that molecular chemoprevention of morin targets NF-κB and acts as a potent anti-inflammatory and pro-apoptotic agent for colon cancer prevention.

Protective effect of p-coumaric acid against 1,2 dimethylhydrazine induced colonic preneoplastic lesions in experimental rats.

Oxidative stress and gut microbial enzymes are intricately linked to the onset of colon carcinogenesis. Phytochemicals that modulate these two factors hold promise for the development of such agents as anticancer drugs. The present study evaluates the chemopreventive potential of p-coumaric acid (p-CA) - a phenolic acid in rats challenged with the colon specific procarcinogen DMH (1,2 di-methyl hydrazine). Rats were randomized into six groups (n=7/group). Group 1 (control); Group 2 (p-CA 200mg/kg b.w.); Group 3 (DMH 40mg/kg b.w.); Groups 4 (DMH+p-CA 50mg/kg b.w.) and Group 5 (DMH+p-CA 100mg/kg b.w.) and Group 6 (DMH+p-CA 200mg/kg b.w.). After the experimental duration of 15 weeks' rats were subjected to necropsy and tissues were collected for the histological and biochemical investigations. DMH induced colonic preneoplastic lesions viz., aberrant crypt foci (ACF), dysplastic ACF (DACF), mucin depleted foci (MDF) and beta catenin accumulated crypts (BCAC) were significantly suppressed by p-CA supplementation. Glucuronide conjugation of DMH in liver and its subsequent deconjugation mediated by microbes in the colon induced the formation of colonic preneoplastic lesions. p-CA inhibited these lesions and protected the rat colon against genotoxic insult by scavenging the free radicals via its strong antioxidant response and detoxification mechanism as measured by TBARS and enzymic antioxidants in control and experimental rats. Of the three tested doses, p-CA at a dose of 100mg/kg body weight is found to exhibit a significant optimum effect compared to the other two doses 50mg/kg body weight and 200mg/kg body weight.

Vasorelaxant and cardiovascular properties of the essential oil of Pogostemon elsholtzioides.

ETHNOBOTANICAL RELEVANCE: Pogostemon elsholtzioides Benth. (Lamiaceae) is an aromatic shrub, endemic to eastern Himalaya region. The leaves are used for treating goiter and high blood pressure (BP) by indigenous people in Arunachal Pradesh, India. Young leaves are used as vegetable and leaf decoction is also used for cough, cold and headache by some indigenous communities in Northeast India. AIM OF THE STUDY: This species is used for treating hypertension and the genus Pogostemon is rich in essential oil. Therefore, the present study was aimed at investigation of the chemical constituents, vasorelaxant and cardiovascular effects of the essential oil of P. elsholtzioides. MATERIALS AND METHODS: P. elsholtzioides was collected from Pasighat, Arunachal Pradesh, India and essential oil was extracted from shade dried leaves. Essential oil was analyzed by GC-FID and GC-MS and the volatile constituents were identified. Vasorelaxant and cardiovascular properties of the essential oil were studied against phenylephrine induced contraction in isolated endothelium intact aortic preparations and by measuring systolic and diastolic BP, mean arterial pressure (MAP) and heart rate (HR) after carotid artery cannulation in Wistar rats. RESULTS: The essential oil was rich in sesquiterpenes and curzerene, benzophenone, α-cadinol and germacrone were major constituents. The essential oil exhibited significant vasodilation effect in phenylephrine induced contracted aortic rings. Vasorelaxant effect of the essential oil was also observed both in the presence and absence of Nitro-L-arginine methyl ester against phenylephrine-contracted aortic rings. It also induced reduction of systolic and diastolic BP, MAP and HR. CONCLUSIONS: Essential oil of P. elsholtzioides exhibited significant vasorelaxant effect against endothelium intact aortic preparation mediated through nitric oxide dependent pathway and also reduced BP. However, further study is needed to screen the role of calcium ions in both intracellular and extracellular pathway.

Morin and Esculetin supplementation modulates c-myc induced energy metabolism and attenuates neoplastic changes in rats challenged with the procarcinogen 1,2 - dimethylhydrazine.

Targeting tumor metabolism by natural products is a novel approach and provides rationale for anti-cancer drug discovery. The present study aims to explore the impact of morin and/or esculetin on c-myc induced energy metabolism in 1,2-dimethylhydrazine (DMH) induced colon cancer in rats. In order to achieve this aim we analyzed the expression of glucose and glutamine transporters and the key enzymes of glycolytic pathway besides the markers of neoplastic changes viz., mucin depleted foci (MDF), beta catenin accumulated crypts (BCAC), and markers of cell proliferation viz., proliferating cell nuclear antigen (PCNA), argyrophilic nucleolar antigen (AgNOR), c-myc, c-jun and c-fos. All the parameters tested in the present study are highly influenced by the phytochemicals morin and/or esculetin in a way to prevent colon carcinogenesis. Morin and/or esculetin supplementation effectively targets tumor metabolism via ß-cateinin/c-myc signaling and affects glycolysis and glutaminolysis to abrogate colon cancer in rats. The anti-cancer effect of morin is more pronounced than esculetin. The effect obtained through the combined treatment of morin and esculetin is comparable to that of individual supplementation of morin and there is no synergistic effect. Overall individual supplementation of morin scores well as a potential anticancer agent targeting glycolysis and glutaminolysis in colon cancer.

Multi-functional nanoparticles as theranostic agents for the treatment & imaging of pancreatic cancer.

Theranostics has received considerable attention since both therapy and imaging modalities can be integrated into a single nanocarrier. In this study, fluorescent iron oxide (FIO) nanoparticles and gemcitabine (G) encapsulated poly(lactide-co-glycolide) (PLGA) nanospheres (PGFIO) conjugated with human epidermal growth factor receptor 2, (HER-2) antibody (HER-PGFIO) were prepared and characterized. HER-PGFIO showed the magnetic moment of 10emu/g, relaxivity (r2) of 773mM-1s-1 and specific absorption rate (SAR) of 183W/g. HER-PGFIO showed a sustained release of gemcitabine for 11days in PBS (pH 7.4). In vitro cytotoxicity evaluation of HER-PGFIO in 3D MIAPaCa-2 cultures showed 50% inhibitory concentration (IC50) of 0.11mg/mL. Subcutaneous tumor xenografts of MIAPaCa-2 in SCID mice were developed and the tumor regression study at the end of 30days showed significant tumor regression (86±3%) in the HER-PGFIO with magnetic hyperthermia (MHT) treatment group compared to control group. In vivo MRI imaging showed the enhanced contrast in HER-PGFIO+MHT treated group compared to control. HER-PGFIO showed significant tumor regression and enhanced MRI in treatment groups, which could be an effective nanocarrier system for the treatment of pancreatic cancer. STATEMENT OF SIGNIFICANCE: Combination therapies are best suitable to treat pancreatic cancer. Theranostics are the next generation therapeutics with both imaging and treatment agents encapsulated in a single nanocarrier. The novelty of the present work is the development of targeted nanocarrier that provides chemotherapy, thermotherapy and MRI imaging properties. The present work is the next step in developing the nanocarriers for pancreatic cancer treatment. Different treatment modalities embedding into a single nanocarrier is the biggest challenge that was achieved without compromising the functionality of each other. The surface modification of polymeric nanocarriers for antibody binding and their multifunctional abilities will appeal to wider audience.

Gastroprotective potential of hydro-alcoholic extract of Pattanga (Caesalpinia sappan Linn.).

ETHNO-PHARMACOLOGICAL RELEVANCE: Pattanga is botanically equated as Caesalpinia sappan Linn. (Family: Caesalpiniaceae) and is used in Ayurveda system of medicine since ages. According to Ayurveda, useful part is Heartwood, which is bitter, astringent and acrid and is useful in vitiated conditions of vata and pitta, burning sensation, wounds, ulcers, leprosy, skin diseases, menorrhagia, leucorrhea, and diabetes. It is used as a major ingredient in Ayurvedic formulations and preparations like Patrangasava, Chandanadya Thalia, and Karpuradyarka. AIM OF THE STUDY: The present study is planned to evaluate the gastroprotective activity of the selected Ayurvedic drug using three different in vivo gastric ulcer models, so as to provide scientific evidence for the Ayurvedic claims. MATERIALS AND METHODS: For this study, Wistar albino rats fasted overnight were selected. The hydroalcoholic extract of Caesalpinia sappan heartwood at the dose level 250 and 500mg/kg body weight was selected and administered orally before necrotizing agents. Antioxidant and antiulcer parameters were evaluated and the stomach samples were subjected for histopathological studies. In addition, PGE2 estimation and protein expressions of COX-1, COX-2 and iNOS were analyzed by Western blot. The plant extract was subjected to LCMS/MS analysis. In addition, Cytoprotective effect in isolated gastric mucosal cells, TUNEL Assay, Acid neutralizing capacity assay, H+/K+ ATPase inhibitory assay were performed. RESULTS: The ulcer protection was found to be 92%, 86% and 64% against ethanol, NSAID and pylorus ligation induced ulcer respectively. The hydro-alcoholic extract of C. sappan heartwood exhibited cytoprotective effect with 76.82% reduction against indomethacin-induced cytotoxicity at the concentration of 25µg/ml. C. sappan showed 63.91% inhibition in H+/K+ ATPase inhibitory assay at the concentration 500µg/ml. CONCLUSIONS: Our results depict that Caesalpinia sappan heartwood possesses gastroprotective activity, possibly mediated through cytoprotection and antioxidant mechanisms. The data obtained in the present study provides scientific support for the traditional use of Caesalpinia sappan in the management of peptic ulcer.

Pharmacognostical, antioxidant and antiulcer screening of Cyclea peltata roots

Cyclea peltata Hook. f. & Thomson, belongs to the family Menispermaceae commonly known as "paatha", is a climbing shrub found throughout South and East India. In Indian traditional medicine the tuberous roots are also used to treat jaundice, stomachache, fever and asthma. This study deals with the microscopic study of leafs and roots of C. peltata, along with the physico-chemical, preliminary phytochemical analyses and antioxidant properties. Acute oral toxicity study was conducted as per OECD-423 guidelines and the extract was found to be devoid of any conspicuous acute toxicity in extract treated animals and no mortality upto 2 g/kg by oral route. Hydroalcoholic extract of C. peltata in a dose dependant manner (125 and 250 mg/kg. p.o.) showed significant gastric protection against the ethanol-induced gastric ulcer model in rats.

Antioxidant activity of Nelumbo nucifera (Gaertn) flowers in isolated perfused rat kidney / Atividade antioxidante das flores de Nelumbo nucifera (Gaertn) em rim isolado perfundido de rato

A padronização da indução de estresse oxidativo com a mstura de Fenton em rim isolado perfundido de rato e a avaliação do efeito antioxidante das flores de Nelumbo nucifera em rim isolado de rato oxidativamente estressado são realizadas no presente estudo. Seis grupos de rim isolado perfundido de rato foram utilizados para o presente estudo. O grupo I de rins recebeu veículo. O grupo II de rins foi oxidativamente estressados com a mistura de Fenton. Os grupos III e IV de animais foram tratados com duas doses graduais de extrato após a administração da mistura de Fenton. Os grupos V e VI de rins foram perfundidos com duas doses graduais de extrato sem a mistura de Fenton. A mistura de Fenton causou estresse oxidativo com diferença significativa de malondialdeído (TBARS), glutationa reduzida (GSH), glutationa peroxidase (GPx), catalase, glutamato oxaloacetato transaminase (GOT) e glutamato piruvato transaminase (GPT) (p<0,05) em ratos doentes. No tratamento dos animais com extrato, o estresse oxidativo foi diminuído com aumento nos antioxidantes e as enzimas marcadoras mantiveram o nível normal. Este efeito do extrato foi dependente da dose. Em conclusão, a mistura de Fenton desenvolveu o estresse oxidativo em rim isolado perfundido de rato e o extrato das flores de Nelumbo nucifera apresenta atividade antioxidante no rim isolado perfundido oxidativamente estressado.

Standardization of induction of oxidative stress with Fenton mixture in isolated perfused rat kidney and evaluation of antioxidant effect of Nelumbo nucifera flowers in isolated oxidatively stressed rat kidney are carried out in present study. Six groups of isolated perfused rat kidney were used for the present study. Group I kidneys were received vehicle. Group II kidneys were oxidatively stressed with Fenton mixture. Group III and IV animals were treated with two graded doses of extract after the administration of Fenton mixture. Group V and VI kidneys were perfused with two graded doses of extract without Fenton mixture. Fenton mixture was found to cause oxidative stress with significant difference of malondialdehyde (TBARS), Reduced glutathione (GSH), Glutathione peroxidase (GPx), catalase, Glutamate Oxaloacetate Transaminase (GOT) and Glutamate Pyruvate Transaminase (GPT) (p<0.05) in diseased rats. On treating animals with extract, the oxidative stress was decreased with increase in antioxidants and the marker enzymes were found to maintain the normal level. This effect of extract was found to be dose dependent. In conclusion, Fenton mixture developed the oxidative stress in isolated perfused rat kidney and Nelumbo nucifera flowers extract exhibits antioxidant activity in that oxidatively stressed isolated perfused kidney.