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BACKGROUND: The Transmembrane Serine Protease 2 (TMPRSS2) of human cell plays a significant role in proteolytic cleavage of SARS-Cov-2 coronavirus spike protein and subsequent priming to the receptor ACE2. Approaching TMPRSS2 as a therapeutic target for the inhibition of SARS-Cov-2 infection is highly promising. Hence, in the present study, we docked the binding efficacy of ten naturally available phyto compounds with known anti-viral potential with TMPRSS2. The aim is to identify the best phyto compound with a high functional affinity towards the active site of the TMPRSS2 with the aid of two different docking software. Molecular Dynamic Simulations were performed to analyse the conformational space of the binding pocket of the target protein with selected molecules. RESULTS: Docking analysis using PyRx version 0.8 along with AutoDockVina reveals that among the screened phyto compounds, Genistein shows the maximum binding affinity towards the hydrophobic substrate-binding site of TMPRSS2 with three hydrogen bonds interaction ( - 7.5 kcal/mol). On the other hand, molecular docking analysis using Schrodinger identified Quercetin as the most potent phyto compound with a maximum binding affinity towards the hydrophilic catalytic site of TMPRSS2 ( - 7.847 kcal/mol) with three hydrogen bonds interaction. The molecular dynamics simulation reveals that the Quercetin-TMPRSS complex is stable until 50 ns and forms stable interaction with the protein ( - 22.37 kcal/mol of MM-PBSA binding free energy). Genistein creates a weak interaction with the loop residues and hence has an unstable binding and exits from the binding pocket. CONCLUSION: The compounds, Quercetin and Genistein, can inhibit the TMPRSS2 guided priming of the spike protein. The compounds could reduce the interaction of the host cell with the type I transmembrane glycoprotein to prevent the entry of the virus. The critical finding is that compared to Genistein, Quercetin exhibits higher binding affinity with the catalytic unit of TMPRSS2 and forms a stable complex with the target. Thus, enhancing our innate immunity by consuming foods rich in Quercetin and Genistein or developing a novel drug in the combination of Quercetin and Genistein could be the brilliant choices to prevent SARS-Cov-2 infection when we consider the present chaos associated with vaccines and anti-viral medicines.
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Tratamento Farmacológico da COVID-19 , Glicoproteína da Espícula de Coronavírus , Antivirais/farmacologia , Genisteína/farmacologia , Humanos , Simulação de Acoplamento Molecular , Quercetina/farmacologia , SARS-CoV-2 , Serina Endopeptidases , Internalização do VírusRESUMO
INTRODUCTION: The present study aimed to realize human recombinant leptin 's ability to synthesize VEGF A while inducing neovascularization through PI3K/Akt/mTOR/S6 kinase involved signaling pathway. METHODS: To examine the PI3K/Akt/mTOR/S6 kinase pathway involvement in leptin-induced VEGF A synthesis, the chick chorioallantoic membrane (CAM) was incubated with human recombinant leptin and specific inhibitors of the proposed signaling molecules (rapamycin and wortmannin). We analyzed the role of specified signaling molecules in human recombinant leptin-induced physiological angiogenesis via VEGF A synthesis in detail with the support of various methodologies. RESULTS: Human recombinant leptin's ability to synthesize VEGF A is diminished significantly in the presence of inhibitors. This observation supported the role of PI3K/Akt/mTOR/S6 kinase signaling molecules in human recombinant leptin-mediated VEGF A synthesis while inducing angiogenesis in CAM. CONCLUSION: Synthesis of VEGF A, followed by the growth of new blood vessels, by human recombinant leptin via the activation of the PI3K/Akt/mTOR/S6 kinase signaling pathway reflects mechanistic therapeutic application of human recombinant leptin. The data also signify the role of mTOR and S6 kinase molecules in angiogenesis under a physiological environment.
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Indutores da Angiogênese/farmacologia , Vasos Sanguíneos/efeitos dos fármacos , Membrana Corioalantoide/irrigação sanguínea , Leptina/farmacologia , Neovascularização Fisiológica/efeitos dos fármacos , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Quinases S6 Ribossômicas/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Fator A de Crescimento do Endotélio Vascular/biossíntese , Animais , Vasos Sanguíneos/enzimologia , Embrião de Galinha , Desenvolvimento Embrionário/efeitos dos fármacos , Humanos , Proteínas Recombinantes/farmacologia , Transdução de Sinais , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Vasodilation occurs as a result of the relaxation of the smooth muscle cells present in the walls of blood vessels. Various suitable models are available for the analysis of the vasoactive properties of drugs with therapeutic applications. But all these models have limitations, such as ethical issues and high cost. The purpose of this study is to develop an alternative model for studying the vasoactive properties of drugs using an in-ovo chicken embryo model. In the preliminary experiment, we used a well-known vasoconstrictor (adrenaline) and a vasodilator (spermine NoNoate) in the chick embryo area vasculosa and evaluated their concentration-response curve. Adrenaline (10 µM) and spermine NoNoate (10 µM) were administered in different arteries and veins and different positions of the right vitelline artery of the chick embryo. Results showed the middle of the vessel bed of the right vitelline artery having the best vasoactive effect compared to others. Finally, anti-hypertensive drugs, calcium channel blockers, and NOS agonists were administered in the chick embryo area vasculosa to validate the model. Results demonstrate that the chick embryo area vasculosa can be an alternative, robust, and unique in-ovo model for screening of anti-hypertensive drugs in real time.
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Avaliação Pré-Clínica de Medicamentos/métodos , Microscopia Intravital/métodos , Vasoconstritores/isolamento & purificação , Vasoconstritores/farmacologia , Vasodilatadores/isolamento & purificação , Vasodilatadores/farmacologia , Animais , Embrião de GalinhaRESUMO
BACKGROUND: Osteosarcoma is the most common primary tumor that affects usually children. Due to its cellular complex and osteoid formation it is very difficult to understand the mechanism behind the progressiveness of osteosarcoma. Various animal models are available to study the issue but they are time consuming and costly. We aimed to understand the progressiveness and invasiveness of osteosarcoma induced by SaOS2 cells using chicken chorioallantoic membrane. CAM is a well-established model which allows in vivo studies of tumor induced angiogenesis and the testing of anti angiogenic molecules. However only a few reports showed the tumor forming ability of SaOS2 cells on CAM. METHOD: Angiogenic ability of SaOS2 cells on CAM was validated by various methods. Angiogenic ability was scored by direct visualization and scanning microscopic analysis. The sprouting ability and growth of the vessel was measured by Angioquant software under different cellular volume. The invasiveness was analyzed by histological staining. Involvement of angiogenic factors at differential stage of progressiveness was confirmed by the molecular and protein level expression analysis. RESULT: SaOS2 cells induces sprouting angiogenesis on CAM and shows its aggressiveness by rupturing the ectodermal layer of the CAM. Growth and development of osteosarcoma depends mainly on the activation of VEGF165, MMP2 and MMP9. CAM able to reproduce angiogenic response against the stimulation of SaOS2 cells exactly as in other animal models without inflammatory reactions. CONCLUSION: CAM is an excellent alternative in vivo model for studying the aggressiveness and tumor progression of osteosarcoma using various angiogenic techniques in an easily, faster and affordable way. We further provided insight about the involvement of various angiogenic growth factors on the development of osteosarcoma which will enable to find the suitable therapeutic molecule for the treatment of osteosarcoma. CAM model could provide a wide space using modern techniques like micro array or in situ hybridization to have a better understanding about the progression and invasiveness of osteosarcoma cells to develop suitable therapeutic molecules.
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BACKGROUND: Leptin, the cytokine produced by white adipose tissue is known to regulate food energy homeostasis through its hypothalamic receptor. In vitro studies have demonstrated that leptin plays a major role in angiogenesis through binding to the receptor Ob-R present on ECs by stimulating and initiating new capillary like structures from ECs. Various in vivo studies indicate that leptin has diverse effect on angiogenesis. A few reports have showed that leptin exerts pro angiogenic effects while some suggested that it has antiangiogenic potential. It is theoretically highly important to understand the effect of leptin on angiogenesis to use as a therapeutic molecule in various angiogenesis related pathological conditions. Chicken chorio allantoic membrane (CAM) on 9th day of incubation was incubated with 1, 3 and 5 µg concentration of HRL for 72 h using gelatin sponge. Images where taken after every 24 h of incubation and analysed with Angioguant software. The treated area was observed under microscope and histological evaluation was performed for the same. Tissue thickness was calculated morphometrically from haematoxylin and eosin stained cross sections. Reverse transcriptase PCR and immunohistochemistry were also performed to study the gene and protein level expression of angiogenic molecules. RESULTS: HRL has the ability to induce new vessel formation at the treated area and growth of the newly formed vessels and cellular morphological changes occur in a dose dependent manner. Increase in the tissue thickness at the treated area is suggestive of initiation of new capillary like structures. Elevated mRNA and protein level expression of VEGF165 and MMP2 along with the activation of ECs as demonstrated by the presence of CD34 expression supports the neovascularization potential of HRL. CONCLUSION: Angiogenic potential of HRL depends on the concentration and time of incubation and is involved in the activation of ECs along with the major interaction of VEGF 165 and MMP2. It is also observed that 3 µg of HRL exhibits maximum angiogenic potential at 72 h of incubation. Thus our data suggest that dose dependent angiogenic potential HRL could provide a novel role in angiogenic dependent therapeutics such as ischemia and wound healing conditions.
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Indutores da Angiogênese/administração & dosagem , Membrana Corioalantoide/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Leptina/administração & dosagem , Neovascularização Fisiológica/efeitos dos fármacos , Zigoto , Animais , Antígenos CD34/metabolismo , Embrião de Galinha , Membrana Corioalantoide/irrigação sanguínea , Membrana Corioalantoide/enzimologia , Relação Dose-Resposta a Droga , Gelatinases/metabolismo , Humanos , Imuno-Histoquímica , Metaloproteinase 2 da Matriz/metabolismo , Microscopia , RNA Mensageiro/metabolismo , Proteínas Recombinantes/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Progression of liver fibrosis to HCC (hepatocellular carcinoma) is a very complex process which involves several pathological phenomena, including hepatic stellate cell activation, inflammation, fibrosis and angiogenesis. Therefore inhibiting multiple pathological processes using a single drug can be an effective choice to curb the progression of HCC. In the present study, we used the mTOR inhibitor everolimus to observe its effect on the in vitro activation of hepatic stellate cells and angiogenesis. The results of the present study demonstrated that everolimus treatment blocked the functions of the immortalized human activated hepatic stellate cell line LX-2 without affecting the viability and migration of primary human stellate cells. We also observed that treatment with everolimus (20 nM) inhibited collagen production by activated stellate cells, as well as cell contraction. Everolimus treatment was also able to attenuate the activation of primary stellate cells to their activated form. Angiogenesis studies showed that everolimus blocked angiogenesis in a rat aortic ring assay and inhibited the tube formation and migration of liver sinusoidal endothelial cells. Finally, everolimus treatment reduced the load of tumoral myofibroblasts in a rat model of HCC. These data suggest that everolimus targets multiple mechanisms, making it a potent blocker of the progression of HCC from liver fibrosis.
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Inibidores da Angiogênese/farmacologia , Células Estreladas do Fígado/efeitos dos fármacos , Imunossupressores/farmacologia , Sirolimo/análogos & derivados , Actinas/metabolismo , Animais , Carcinoma Hepatocelular/tratamento farmacológico , Diferenciação Celular , Linhagem Celular Tumoral , Movimento Celular , Sobrevivência Celular , Colágeno/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Everolimo , Humanos , Fígado/efeitos dos fármacos , Cirrose Hepática/patologia , Neoplasias Hepáticas/tratamento farmacológico , Masculino , Músculo Liso/metabolismo , Miofibroblastos/citologia , Miofibroblastos/metabolismo , Transplante de Neoplasias , Neovascularização Patológica , Ratos , Ratos Wistar , Sirolimo/farmacologiaRESUMO
Background: High-molecular weight heparin (HMWH), a molecule extensively used as an anticoagulant, shows concentration-dependent angiogenic and anti-angiogenic potential. So far, no studies have reported the interactive potential of HMWH with various pro-angiogenic growth factors under physiological conditions. Haence, we aimed to find the impact of major pro-angiogenic growth factors under HMWH induced angiogenesis. Methods: Chicken Chorioallantoic Membranes (CAMs) are incubated with various concentrations of HMWH. Semiquantitative PCR method was implemented to measure the changes in the transcription level of pro-angiogenic growth factors. The scanning electron microscopic technique is applied to find the morphological changes in CAM. Molecular docking and molecular dynamics simulation studies using NAMD and CHARMM force field discerned the heparin-binding mode with the pro-angiogenic growth factors. Results: HMWH can enhance the transcription level of major pro-angiogenic growth factors, significantly impacting FGF2 under 100 µM concentration. The in-silico analysis reveals that HMWH shows the highest binding affinity with FGF2. Further, molecular dynamics and interaction studies using 1 kDa Heparin against FGF2 showed that the former binds stably with the latter due to a strong salt bridge formation between the sulfate groups and arginine residues (ARG 119 and ARG109). Conclusion: The combined experimental and in-silico analysis results reveal that HMWH can interact with pro-angiogenic growth factors under micromolar concentration while inducing angiogenesis. This observation further supports the therapeutic benefits of HMWH as an angiogenic factor under such low concentration. This technique is used to replenish the blood supply to chronic wounds to speed healing and prevent unnecessary amputations.
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Exploration of new strategies and identification of less expensive novel chemoprevention agents against breast cancer progression have become the need of the hour. Thus, the present study aimed at evaluating the anti-cancer efficacies of octyl gallate (OG) and gallic acid (GA) isolated from Terminalia bellirica (T. bellirica) in breast cancer cell lines and DMBA-induced Sprague-Dawley animal model. The results of western blot analysis show significant (p < 0.05) downregulation of anti-apoptotic protein (Bcl-2 and Bcl-xL) expression and up-regulation of pro-apoptotic protein (Bak and Bax) expression in both MCF-7 and MDA-MB-231 cell lines. Our findings also show that DMBA-induced Sprague-Dawley rats (50-55 days old) orally administered with OG (20 mg/kg body wt.) and GA (20 mg/kg body wt.) for a treatment period of 14 weeks were observed for normalized body weight changes and hematological indices and significant reduction of tumor markers carcinoembryonic antigen (CEA), cancer antigen 15.3 (CA 15.3), and oxidative stress (TBARS) in serum, while the activity of anti-oxidant enzyme (SOD, CAT, and GPx) levels estimated in the mammary tissue was found restored back to normal. Computational molecular interaction study was also performed to substantiate the in vitro obtained results. The tissue histology reveals the therapeutic role of OG and GA. The study conducted brings to limelight of the molecular mechanisms of intrinsic apoptotic signaling pathway through which OG and GA exert their chemopreventive action. Both OG and GA can be explored further as chemotherapeutic natural drugs for their ability to prevent breast cancer progression.
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Neoplasias , Terminalia , Ratos , Animais , Antioxidantes/farmacologia , Ratos Sprague-Dawley , Transdução de Sinais , Ácido Gálico/farmacologia , ApoptoseRESUMO
The study aims to identify and validate a potential α-Amylase inhibitor from the leaf extract of the Parthenium hysterophorus. Molecular docking and dynamics analyses were performed to test the anti-diabetic efficacy of the compound by focusing on α-Amylase inhibition. The molecular docking study using AutoDock Vina (PyRx) and SeeSAR tools identified ß-Sitosterol as an effective α-Amylase inhibitory compound. Among the analysed fifteen phytochemicals, ß-Sitosterol demonstrated the most appreciable binding energy (-9.0 Kcal/mol) and is comparatively higher than the binding energy of the standard α-Amylase inhibitor, the Acarbose (-7.6 Kcal/mol). The significance of the interaction between ß-Sitosterol and α-Amylase was further investigated using Molecular Dynamics Simulation (MDS) for 100 ns via GROMACS. The data reveals that the compound could exhibit the highest stability with α-Amylase regarding RMSD, RMSF, SASA and Potential Energy analysis. The key residue of α-Amylase (Asp -197) shows a significantly low fluctuation of 0.7 Å while interacting with ß-Sitosterol. The data obtained from MDS results strongly suggested the potential inhibitory impact of ß-Sitosterol on α-Amylase. In addition, the proposed phytochemical was purified from the leaf extracts of P.hysterophorus using the silica gel column chromatography and identified by GC-MS analysis. The purified ß-Sitosterol demonstrated a significant 42.30% in-vitro α-Amylase enzyme inhibition property under 400 µg/ml concentration and thus supported the in-silico predictions. Further in-vivo investigations are necessary to analyse the efficiency of ß-Sitosterol on α-Amylase inhibition to help the anti-diabetic potential of the phytocompound.Communicated by Ramaswamy H. Sarma.
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Parthenium hysterophorus , alfa-Amilases , Simulação de Acoplamento Molecular , Extratos Vegetais/farmacologia , Extratos Vegetais/químicaRESUMO
Aim: Multiple parameters are available to predict the outcome of critically sick neonates admitted in neonatal intensive care unit (NICU). Main aim of the study is to validate the role of TOPS, especially the post-transport TOPS score as a simplified assessment of neonatal acute physiology in predicting mortality among transported neonates admitted at level III NICU. Also, to compare the efficiency of post transport TOPS score with SNAP II PE in predicting mortality. Methods: A prospective study carried out with 85 neonates transported from various primary health care centres to level III NICU. Physiological status of the neonates was assessed with the help of pre and post transport TOPS scores. Post-transport TOPS score was recorded immediately after the admission and SNAP II PE within 24 h of admission at level III NICU. Receiver operating characteristics analysis was performed to observe the mortality prediction efficiency of TOPS score and was compared with SNAP II PE. Results: 64 neonates were died due to asphyxia and preterm birth (32%) related complications. Strong significant association with the mortality rate was found between the total post transport TOPS score (0.001) and SNAP II PE (0.003). The AUC, sensitivity and specificity of post transport TOPS score for a cut-off value ≤7 were 0.900, 87.5% and 80% and significant (<0.001) and for SNAP II PE for a cut-off value >12 were 0.913, 75.5% and 100% and is significant (<0.001). Conclusion: TOPS score, especially the post transport TOPS score has an equally good prediction capacity of mortality similar like SNAP II PE among mobilised critically ill neonates. Hence, the TOPS score can be used as a simple and effective method to predict mortality risk among transported neonates immediately after admission at level III NICU.
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Introduction Cervical cancer is the fourth most frequent cancer in women worldwide, and it continues to be a big issue in developing countries. The current case-control study sought to determine the presence of high-risk human papillomaviruses (hr-HPV) in the development of cervical cancer, as well as their relationship with the cell cycle inhibitor gene p16INK4A in cervical cancer. Methods The association between p16INK4A protein and the presence of hr-HPV DNA in cervical lesions was explored in this study, which included 150 cervical cancer patients and 100 normal cervix samples. The immunohistochemistry approach was used to identify the expression of the p16INK4A protein, while the semi-quantitative polymerized chain reaction (PCR) method was used to identify the genomic identity of hr-HPV. Results About 90.67% (n=136) of the 150 case samples were found to be hr-HPV positive. Within the 136 HPV-positive samples, 45 (33.08%) show moderate expression of the p16INK4A protein, whereas 91 (66.91%) show overexpression, which is statistically significant (0.05). Among the 136 HPV-positive samples, 22.08% (N=30) were classified as having cervical intraepithelial neoplasia (CIN), with 56.66% (n=17) having CIN3, 36.66% (n=11) having CIN2, and 6.67% (n=2) having CIN1. Conclusion Based on the semi-quantitative immune staining scoring method of p16INK4A protein, genomic expression of HPV demonstrates that the expression of p16INK4A protein increases with the infectious load of the hr-HPV genome in the host cell. The result directly shows that immunostaining of the p16INK4A protein, in conjunction with the assessment of high-risk HPV in the host genome, will aid in the identification of cervical cancer in the cervix.
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Cisplatin is the most commonly used first-line drug for cancer treatment. However, many patients develop resistance to cisplatin therapy which ultimately results in therapy failure and increased mortality. A growing body of evidence shows that the hypoxic microenvironment is the prime factor underlying tumor insensitivity to cisplatin treatment. Since tumors in the majority of cancer patients are under hypoxic stress (low oxygen supply), it becomes necessary to understand the pathobiology behind hypoxia-induced cisplatin resistance in cancer cells. Here, we discuss the molecular events that render hypoxic tumors insensitive to cisplatin therapy. Furthermore, various drugs and tumor oxygenation techniques have been developed to circumvent cisplatin resistance in hypoxic tumors. However, their pharmaceutical applications are limited due to failures in clinical investigations and a lack of preclinical studies in the hypoxic tumor microenvironment. This review addresses these challenges and provides new directions for the strategic deployment of cisplatin sensitizers in the hypoxic tumor microenvironment.
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Cisplatino , Neoplasias , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Humanos , Hipóxia , Neoplasias/tratamento farmacológico , Hipóxia Tumoral , Microambiente TumoralRESUMO
BACKGROUND: The study's main aim is to compare and correlate the levels of various hematological indices in type 2 DM patients with the gender, age, duration, and family history of diabetic conditions to predict diabetes-related complications. METHODS: The diabetic population is divided into 2, group 1- subjects with complications and group 2- subjects without complications. Hematological indices are measured using an automated analyzer. RESULTS: Females from group 1 show a significantly higher value for PLC (3.72 ± 4.79/<0.05) and positively correlate with the diabetic duration. Females with >40 years of age from group 2 show a significantly higher value for platelet larger cell ratio (P-LCR/%) (40.17 ± 3.25 (>40)/<0.05) than those with <40 age and positively correlated with the age. Males with >40 years of age from group 1 show a significantly higher value for plateletcrit (PCT/%) (0.297 ± 0.067 (>40)/<0.05) than those from <40 age and positively correlated with the age. All the male subjects show significant higher values for hemoglobin concentration (HB/g/dl) (13.49 ± 2.22)/<0.05 for group 1) (13.61 ± 2.02)/<0.05 for group 2) and hematocrit (HCT/L/L) (37.30 ± 7.55/<0.05 for group 1) (38.64 ± 5.42/<0.05 for group 2). CONCLUSION: Correlating the hematological indices with the gender, age, and duration of diabetic condition will help determine future complications and the severity of the diabetic condition in type 2 DM patients.
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Diabetes is one of the most significant health issues across the world. People identified with diabetes are more vulnerable to various infections and are at a greater risk of developing cardiovascular diseases. The plant-based food we consume often contains many sterol-based bioactive compounds. It is well documented that these compounds could effectively manage the processes of insulin metabolism and cholesterol regulation. Insulin resistance followed by hyperglycemia often results in oxidative stress level enhancement and increased reactive oxygen species production. At the molecular level, these changes induce apoptosis in pancreatic cells and hence lead to insulin insufficiency. Studies have proved that plant sterols can lower inflammatory and oxidative stress damage connected with DNA repair mechanisms. The effective forms of phyto compounds are polyphenols, terpenoids, and thiols abundant in vegetables, fruits, nuts, and seeds. The available conventional drug-based therapies for the prevention and management of diabetes are time-consuming, costly, and with life-threatening side effects. Thereby, the therapeutic management of diabetes with plant sterols available in our daily diet is highly welcome as there are no side effects. This review intends to offer an overview of the present scenario of the anti-diabetic compounds from food ingredients towards the therapeutic beneficial against diabetes.
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BACKGROUND: Leptin, the cytokine produced by white adipose tissue is known to regulate food energy homeostasis through its hypothalamic receptor. In vitro studies have demonstrated that leptin plays a major role in angiogenesis through binding to the receptor Ob-R present on ECs by stimulating and initiating new capillary like structures from ECs. Various in vivo studies indicate that leptin has diverse effect on angiogenesis. A few reports have showed that leptin exerts pro angiogenic effects while some suggested that it has antiangiogenic potential. It is theoretically highly important to understand the effect of leptin on angiogenesis to use as a therapeutic molecule in various angiogenesis related pathological conditions. Chicken chorio allantoic membrane (CAM) on 9th day of incubation was incubated with 1, 3 and 5 µg concentration of HRL for 72 h using gelatin sponge. Images where taken after every 24 h of incubation and analysed with Angioguant software. The treated area was observed under microscope and histological evaluation was performed for the same. Tissue thickness was calculated morphometrically from haematoxylin and eosin stained cross sections. Reverse transcriptase PCR and immunohistochemistry were also performed to study the gene and protein level expression of angiogenic molecules. RESULTS: HRL has the ability to induce new vessel formation at the treated area and growth of the newly formed vessels and cellular morphological changes occur in a dose dependent manner. Increase in the tissue thickness at the treated area is suggestive of initiation of new capillary like structures. Elevated mRNA and protein level expression of VEGF165 and MMP2 along with the activation of ECs as demonstrated by the presence of CD34 expression supports the neovascularization potential of HRL. CONCLUSION: Angiogenic potential of HRL depends on the concentration and time of incubation and is involved in the activation of ECs along with the major interaction of VEGF 165 and MMP2. It is also observed that 3 µg of HRL exhibits maximum angiogenic potential at 72 h of incubation. Thus our data suggest that dose dependent angiogenic potential HRL could provide a novel role in angiogenic dependent therapeutics such as ischemia and wound healing conditions.