TXA2 mediates LPA1-stimulated uterine contraction in late pregnant mouse.

Lysophosphatidic acid (LPA) is known to increase uterine contraction in the estrus cycle and early pregnancy, however, the effect of LPA in late pregnant uterus and its mechanisms are not clear. In the present study, we show the LPA receptor subtypes expressed and the mechanism of LPA-induced contractions in late pregnant mouse uterus. We determined the relative mRNA expression of LPA receptor genes by quantitative PCR and elicited log concentration-response curves to oleoyl-L-α-LPA by performing tension experiments in the presence and absence of nonselective and selective receptor antagonists and inhibitors of the TXA2 pathway. LPA1 was the most highly expressed receptor subtype in the late pregnant mouse uterus and LPA1/2/3 agonist (Oleoyl-L-α LPA) elicited increased contractions in this tissue that had lesser efficacy compared to oxytocin. LPA1/3 antagonist, Ki-16425, and a potent LPA1 antagonist (AM-095) significantly inhibited the LPA-induced contractions. Further, the nonselective COX inhibitor, indomethacin, and potent thromboxane A2 synthase inhibitor, furegrelate significantly impaired LPA-induced contractions. Moreover, selective thromboxane receptor (TP) antagonist, SQ-29548, and Rho kinase inhibitor, Y-27632 almost eliminated LPA-induced uterine contractions. LPA1 stimulation elicits contractions in the late pregnant mouse uterus using the contractile prostanoid, TXA2 and may be targeted to induce labor in uterine dysfunctions/ dystocia.

PRE-084 ameliorated kidney injury by reducing endoplasmic reticulum stress in the rat model of adenine-induced chronic kidney disease.

BACKGROUND: Endoplasmic reticulum (ER) stress plays an important role in the development of chronic kidney disease (CKD). Sigma-1 receptors (σ1Rs) are novel chaperone proteins that regulate ER stress. However, effect of σ1R activation on renal ER stress is yet unexplored. So, in the present study we investigated the effects of PRE-084, a σ1R agonist on renal injury and ER stress in the rat model of CKD. METHODS: CKD group rats were fed adenine for 28 days and CKD treatment group rats were additionally administered PRE-084 intraperitoneally at 1, 3 and 10 mg/kg body weight dose from Day 22-28. ER stress markers were evaluated using molecular biology techniques such as immunohistochemistry and Western blot. RESULTS: Marked kidney injury was observed in CKD rats as revealed by biochemical and histological findings. Expression of ER stress proteins such as phosphorylated protein kinase R-like ER kinase (p-PERK), cleaved activating transcription factor-6 (ATF-6f), phosphorylated inositol requiring enzyme1α (p-IRE1α) and caspase-12 were higher in CKD rats. Nevertheless, CKD rats treated with PRE-084 particularly at 10 mg/kg dose showed considerably lesser kidney injury along with higher expression of σ1R and marked reduction of all the ER stress proteins studied. CONCLUSION: Results reveal that PRE-084 likely ameliorated the adenine-induced kidney injury by lowering ER stress through increased σ1R expression.

Biochanin-A alleviates fibrosis and inflammation in cardiac injury in mice.

Biochanin-A (BCA), is an isoflavonoid, exhibits protective effects against various diseases. This study was conducted to observe the effect of BCA on isoprenaline (ISP)-induced cardiac fibrosis and explore the underlying mechanism. The curative effect of BCA was investigated with oral administration for 14 days in ISP-induced cardiac fibrosis in mice. The fibrotic biomarkers, like collagen I and III, were estimated by ELISA. Commercial kits were used to estimate cholesterol, triglycerides, and creatine kinase-myocardial band (CK-MB) levels. The messenger ribonucleic acid (mRNA) expression studies were performed by quantitative real-time polymerase chain reaction. Gelatin zymography was used to study the expression of matrix metalloproteinases-2 (MMP-2). BCA co-administration significantly improved the morphometric parameters; including heart weight, heart weight to body weight, heart weight to tibial length, and lipid profile. BCA treatment showed a reduction in inflammatory cells and collagen deposition as depicted in the histopathology of heart tissues. The enhanced levels of collagen-I, III, and hydroxyproline were significantly decreased by BCA co-treatment, whereas CK-MB level was reduced slightly. BCA co-administration increased the activity of reduced glutathione enzyme, showing the antioxidative effects of BCA. BCA treatment significantly reduced interleukin-6 (Il6) inflammatory cytokine along with partially decreased mRNA expression of fibrotic signaling markers such as natriuretic peptide type B (Nppb), α-smooth muscle actin (Acta2), connective tissue growth factor (Ctgf), transforming growth factor ß (Tgfb), small mothers against decapentaplegic homolog-3 (Smad-3). However, BCA did not modify Mmp-2 expression, which was significantly increased by ISP. In conclusion, BCA exerts an antifibrotic effect by modulating lipid profile, enhancing antioxidant enzyme, and reducing collagen content and inflammation.

Fat augments leptin-induced uterine contractions by decreasing JAK2 and BKCa channel expressions in late pregnant rats.

Altered lipid metabolism in obesity causes pregnancy complications in humans and animals. Leptin levels increase in pregnancy, as well as obesity. However, the effect of obesity on uterine leptin receptors and its distal signaling is not clear. The present study aimed to understand the effect of increased fat on leptin signaling in rat uterus. Wistar female rats were fed with an HF diet (40% Fat, 17% Sucrose, 1.25% Cholesterol, 0.75% Cholic acid) for 6 weeks before the mating and during pregnancy. HF diet significantly increased the fat depots, liver weight, serum, and tissue cholesterol levels. It produced fatty degeneration in the liver and caused infiltration of inflammatory cells, cystic endometrial glands, and sub endometrial fibrosis of the uterus. In isometric tension experiments, leptin caused a significant increase in uterine contractions in high fat-fed animals compared to control animals. Analysis of receptor expressions revealed no significant difference between the groups. However, a significant decrease in the JAK2 and BKCaα mRNA expression was observed in the uterus of high fat-fed rats. No change in the BKCaß, eNOS, iNOS, MLCP, and MLCK mRNA expressions was noticed in the HF group compared to the control. The findings of the present study suggest that the contractile response to leptin in the uterus of high fat-fed rats may be attributed to reduced signaling through JAK2 and, lowered expressions of BKCa channel α subunits.

Subacute 28 days oral toxicity study of kaempferol and biochanin-A in the mouse model.

The present study was undertaken to investigate the safety of kaempferol (KEM) and biochanin-A (BCA) following subacute exposure in mice. KEM and BCA were administered in three different doses by oral administration for 28 days. Evaluation of general toxicity parameters by examining the clinical signs, body weight, organ weights, haematological, biochemical, oxidative stress parameters, and histopathology was done. Administration of KEM and BCA for 28 days did not show any clinical signs of toxicity, nor any treatment-related changes in body weight and organ weights in comparison to control. The haematological parameters such as red blood cell, white blood cell, platelets count, haemoglobin (Hb) level, haematocrit, mean corpuscular haemoglobin concentration, red cell distribution width, and platelet distribution width did not show any change in the treated groups and control. Furthermore, different biochemical parameters like markers of the liver (alanine aminotransferase and aspartate aminotransferase), kidney (creatinine and urea), and heart (creatinine kinase-myocardial band and lactate dehydrogenase) injury along with other biochemical parameters showed nonsignificant differences between treated groups and control. Results of oxidative stress parameters in treated groups showed insignificant variations with control. The level of antioxidant enzymes such as superoxide dismutase and catalase were markedly increased in the treated groups; however, these were nonsignificant in comparison to control. In histopathology, evaluation of all vital organs, such as liver, kidney, heart, and lungs, did not show any morphological abnormalities and lesions in treated groups and control. The present study suggests that KEM and BCA have no adverse effects on the general physiology in mice.

Topical bilirubin-deferoxamine hastens excisional wound healing by modulating inflammation, oxidative stress, angiogenesis, and collagen deposition in diabetic rats.

AIM OF THE STUDY: The study was performed to understand the detailed mechanism of diabetic wound healing by bilirubin-deferoxamine (DFO) combination on topical application. MATERIALS AND METHODS: There were two study groups, control, and treatment. The granulation tissues collected on different days (3, 7, 14, and 19) were studied in detail for inflammatory mediators, angiogenesis markers, epithelialization, and oxidative stress parameters. RESULTS: A significant increase in wound contraction percentage was observed from day 7 in the bilirubin-DFO treatment group. The combinatorial treatment significantly reduced tumour necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1ß), and enhanced IL-10 levels. Upregulated mRNAs of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1 alpha (HIF-1 α) along with CD31 immunohistochemistry showed the pro-angiogenesis potential of the combination. Hematoxylin and Eosin (H and E) staining and Masson's trichrome staining showed reduced inflammatory cell infiltration, enhanced fibroblast proliferation, well-organized collagen fibers, and the development of new blood vessels. Collagen deposition is further supported by immunohistochemistry studies and Masson's trichrome staining. Bilirubin-DFO combination also reduced lipid peroxidation and elevated antioxidative enzymes. CONCLUSION: Topical application of bilirubin-DFO showed immense potential in augmenting skin wound regeneration in diabetes by upregulating the antioxidant status as well as increasing angiogenesis, collagen deposition, and modulating cytokines.

Leptin receptor stimulation in late pregnant mouse uterine tissue inhibits spontaneous contractions by increasing NO and cGMP.

The adipokine, leptin exerts inhibitory effect on both spontaneous and oxytocin-induced contractions in myometrium. However, the mechanisms involved in leptin-induced effect are not clear. In the present study, we studied the altered characteristics of uterine contractions in the presence of leptin and the possible mechanisms of its effect in late pregnant (18.5 day) mouse uterus. We conducted functional, biochemical and molecular biology studies to demonstrate the mechanism of leptin-induced response. Leptin exerted an inhibitory response (Emax 40.5 ± 3.99%) on basal uterine contractions. The extent of inhibition was less than that obtained with known uterine relaxants, salbutamol (Emax103 ± 8.66%) and BRL-37344 (Emax 84.79 ± 8.12%). Leptin-induced uterine response was inhibited by leptin receptor antagonist SHLA and JAK-STAT pathway inhibitor, AG-490. The relaxant response was also subdued by NO-cGMP-PK-G pathway blockers L-NAME, 1400W, ODQ and KT-5823. Further, leptin enhanced the levels of NO and cGMP in uterine tissues. Also, SHLA, AG-490 and a combination of 1400 W and L-NAME prevented leptin-induced increase in NO. Similar effect was observed on cGMP levels in presence of leptin and SHLA. However, leptin did not influence CaCl2-induced response in potassium-depolarized tissues. We also detected leptin receptor protein in late pregnant mouse uterus located in endometrial luminal epithelium and myometrial layers. Real-time PCR studies revealed significantly higher expression of short forms of the receptor (ObRa and ObRc) in comparison to the long form (ObRb). In conclusion, the results of the present study suggest that leptin inhibits mouse uterine contraction by stimulating short forms of the leptin receptors and activating NO pathway in a JAK-STAT-dependent manner.

Hyperlipidemia impairs uterine ß-adrenergic signaling by reducing cAMP in late pregnant rats.

The aim of the present study was to reveal the effect of hyperlipidemia on ß2- and ß3-adrenergic signaling in late pregnant rat uterus. Hyperlipidemia was induced in female Wistar rats by feeding a high-fat high-cholesterol diet for 8 weeks before and after mating upto the 21st day of gestation. The effect of hyperlipidemia on ß-adrenergic signaling was studied with the help of tension experiments, real-time PCR and cAMP ELISA in 21-day pregnant rat uterus. In tension experiments, hyperlipidemia neither altered the spontaneous contractility nor the oxytocin-induced contractions. However, it decreased the -logEC50 values of ß2-adrenoceptor agonist, salbutamol and ß3-adrenoceptor agonist, BRL37344. It also decreased the efficacy of adenylyl cyclase activator, forskolin. Further, there was a significant decrease in salbutamol and BRL37344-stimulated cAMP content in uterine tissues. However, there was no alteration in mRNA expressions of ß2-adrenoceptor (Adrb2), ß3-adrenoceptor (Adrb3) and Gs protein (Gnas) though there was a significant increase in the mRNA expression of Gi protein (Gnai). In conclusion, reduced cAMP content after beta-adrenergic receptor stimulation, which correlates with an increase in Gnai mRNA, may explain the mechanism of the impairment of uterine ß-adrenergic signaling in hyperlipidemic pregnant rats. The clinical implication of the present study may relate to reduced myometrial relaxant response to ß-adrenergic agonists in high fat-induced uterine dysfunction.

Luteolin attenuates acute lung injury in experimental mouse model of sepsis.

The present investigation was undertaken to assess the result of pretreatment of luteolin in sepsis-induced acute lung injury in mice and its mechanism of action. Luteolin was administered intraperitoneally one hour before caecal ligation and puncture (CLP) surgery in mice. Acute lung injury was assessed by estimation of different parameters like lung edema, protein content, cytokines level, oxidative stress, inducible nitric oxide synthase (iNOS), intercellular adhesion molecule (ICAM)-1 expression and histopathology. Pretreatment of mice with luteolin showed decrease lung edema and protein content in tissue and bronchoalveolar lavage fluid (BALF). However, mice pretreated with luteolin showed reduction (p = 0.92) in blood and lung tissue bacterial counts however it was non significant. Further, luteolin showed significant reduction in interleukin (IL)-6 and IL-1ß in lung tissue which are the proinflammatory cytokines. However, plasma IL-1ß and tissue tumor necrosis factor (TNF)-α level decrease (p = 0.24; p = 0.19) with this pretreatment. Further, ICAM-1 mRNA expression and nuclear factor (NF)-kappa B protein expression were significantly (p < 0.01) decreased in luteolin pretreated septic mice. The lung iNOS level, iNOS mRNA and protein expressions were markedly (p = 0.25; p = 0.50; p = 0.06) altered with luteolin pretreatment, respectively. Also, significant reduction in lipid peroxidation and increase in the activity of antioxidant enzymes like superoxide dismutase (SOD) and catalase was noted with luteolin pretreatment. However, luteolin did not alter (p = 0.36) the non enzymatic antioxidant GSH activity in septic mice. Histopathology of lung tissue showed reduction in lung injury with the luteolin pretreatment in septic mice. The study suggests that luteolin showed attenuation in sepsis-induced acute lung injury in mice through suppression in ICAM-1, NF-kappa B, oxidative stress and partially iNOS pathways.

Casein kinase 2 inhibition impairs spontaneous and oxytocin-induced contractions in late pregnant mouse uterus.

NEW FINDINGS: What is the central question of this study? Does the inhibition of the protein kinase casein kinase 2 (CK2) alter the uterine contractility? What is the main finding and its importance? Inhibition of CK2 impaired the spontaneous and oxytocin-induced contractility in late pregnant mouse uterus. This finding suggests that CK2 is a novel pathway mediating oxytocin-induced contractility in the uterus and thus opens up the possibility for this class of drugs to be developed as a new class of tocolytics. ABSTRACT: The protein kinase casein kinase 2 (CK2) is a ubiquitously expressed serine or threonine kinase known to phosphorylate a number of substrates. The aim of this study was to assess the effect of CK2 inhibition on spontaneous and oxytocin-induced uterine contractions in 19 day pregnant mice. The CK2 inhibitor CX-4945 elicited a concentration-dependent relaxation in late pregnant mouse uterus. CX-4945 and another selective CK2 inhibitor, apigenin, also inhibited the oxytocin-induced contractile response in late pregnant uterine tissue. Apigenin also blunted the prostaglandin F2α response, but CX-4945 did not. Casein kinase 2 was located in the lipid raft fractions of the cell membrane, and disruption of lipid rafts was found to reverse its effect. The results of the present study suggest that CK2, located in lipid rafts of the cell membrane, is an active regulator of spontaneous and oxytocin-induced uterine contractions in the late pregnant mouse.

Kaempferol attenuates acute lung injury in caecal ligation and puncture model of sepsis in mice.

AIM OF THE STUDY: Kaempferol is a flavonoid and important part of the diet. Kaempferol has shown antioxidant, antiinflammatory and antidiabetic activities in various studies. However, protective potential of kaempferol in acute lung injury induced by sepsis and its mechanism remains unclear. The present study was undertaken to evaluate the effect of kaempferol in sepsis-induced acute lung injury in mice and its possible mechanism of action. MATERIALS AND METHODS: Acute lung injury was induced by CLP surgery in mice. Kaempferol (100 mg/kg bw) was administered orally one hour before caecal ligation and puncture surgery in mice. Mice were divided into four groups sham, KEM+sham, sepsis (CLP), and KEM+sepsis. Assessment of lung injury was done by estimation of protein content in lung tissue, lung edema, proinflammatory cytokines in plasma and lung tissue, oxidative stress, antioxidant enzymes, nitrite production, and histopathology. RESULTS: Kaempferol pretreated mice showed significant (P < 0.001) decrease in water content in lungs. Kaempferol pretreatment showed reduction in cytokines IL-6, IL-1ß, and TNF-α in plasma as well as in lung tissue in comparison with septic mice without pretreatment. Pretreatment with kaempferol did not show any reduction in MDA level in comparison with septic mice. Antioxidant enzymes SOD and catalase and nonenzymatic antioxidant GSH activities were also increased with kaempferol pretreatment in septic mice. Further, kaempferol pretreatment reduced the lung tissue nitrite level (P < 0.01) and iNOS (P < 0.05) level in septic mice. A significant (P < 0.01) downregulation of mRNA expression of ICAM-1 and iNOS was observed with this pretreatment. Kaempferol pretreatment did not decrease bacterial load in septic mice. Mice pretreated with kaempferol followed by sepsis showed lesser infiltration of cells and more arranged alveolar structure in histopathological analysis. CONCLUSIONS: The study suggests that kaempferol showed attenuation in sepsis-induced acute lung injury in mice through suppression of oxidative stress, iNOS, and ICAM-1 pathways.

Hypercholesterolemia impairs oxytocin-induced uterine contractility in late pregnant mouse.

High cholesterol is known to negatively affect uterine contractility in ex vivo conditions. The aim of the present study was to reveal the effect of in vivo hypercholesterolemia on spontaneous and oxytocin-induced uterine contractility in late pregnant mouse uterus. Female Swiss albino mice were fed with high cholesterol (HC) diet (0.5% sodium cholate, 1.25% cholesterol and 15% fat) for 6 weeks and then throughout the gestation period after mating. On day 19 of gestation, serum cholesterol level was increased more than 3-fold while triglycerides level was reduced in HC diet-fed animals as compared to control animals fed with a standard diet. In tension experiments, neither the mean integral tension of spontaneous contractility nor the response to CaCl2 in high K+-depolarized tissues was altered, but the oxytocin-induced concentration-dependent contractile response in uterine strips was attenuated in hypercholesterolemic mice as compared to control. Similarly, hypercholesterolemia dampened concentration-dependent uterine contractions elicited by a GNAQ protein activator, Pasteurella multocida toxin. However, it had no effect on endogenous oxytocin level either in plasma or in uterine tissue. It also did not affect the prostaglandin release in oxytocin-stimulated tissues. Western blot data showed a significant increase in caveolin-1 and GRK6 proteins but decline in oxytocin receptor, GNAQ and RHOA protein expressions in hypercholesterolemic mouse uterus. The results of the present study suggest that hypercholesterolemia may attenuate the uterotonic action of oxytocin in late pregnancy by causing downregulation of oxytocin receptors and suppressing the signaling efficacy through GNAQ and RHOA proteins.

Involvement of ObRb receptor, nitric oxide, and BKCa channel signaling pathways in leptin-induced relaxation of pregnant mouse uterus.

The purpose of this study was to determine the receptor subtype and the underlying mechanisms involved in the relaxant effect to leptin in mid- and late-pregnant mouse uterus. We determined the relative mRNA expression of receptor subtypes, eNOS, and BKCa channel by quantitative PCR and also the overall receptor expression by immunohistochemistry. Isometric tension studies were conducted to evaluate the effects of leptin and to delineate its mechanisms. A selective siRNA for the ObRb receptor was used to determine the participation of the receptor subtype in biochemical and molecular effects of leptin. The relaxant response to leptin was greater in mid-pregnancy compared to late pregnancy and was mediated by the activation of BKCa channels by eNOS-derived nitric oxide in an ObRb receptor-dependent manner. In comparison to mid-pregnancy, expression of short forms (mainly ObRa receptor) of the receptor was significantly increased in late pregnancy, whereas ObRb receptor expression was similar in both phases. The results of the study suggest that ObRb receptor mediates leptin-induced increase in eNOS expression and NO synthesis. Leptin-induced eNOS expression and activation cause cGMP-independent stimulation of BKCa channels causing uterine relaxation. Increased short forms of the receptors and reduced BKCa channels exert a negative effect on uterine relaxation in late pregnancy. Leptin may have a physiological role in maintaining uterine quiescence in mid-pregnancy and its reduced relaxant response in late gestation may facilitate labor. Further, ObRb receptor agonists may be useful in the management of preterm labor.

Prenatal exposure to ethion caused maternal and foetal toxicity in rats.

Ethion is a class II moderately toxic organothiophosphate pesticide. The main objective of this study was to evaluate the maternal and foetal toxicity of ethion in rats. Pregnant rats were divided into 5 groups. Group I served as control. Group II, III, IV, and V were orally administered with 0.86, 1.71, 3.43, and 6.9 mg/kg of ethion respectively, from gestational day (GD) 6-19. Dams were sacrificed on GD 20. Maternal toxicity was assessed by body weight gain, foetal resorptions, oxidative stress, liver and kidney function tests, and histopathology. Foetal toxicity was assessed by physical status, gross, teratological and histopathological examination. Ethion caused dose-dependent reduction in maternal body weight gain, increased resorptions, and reduced gravid uterine weights. Elevated MDA levels and altered levels of GSH, SOD and catalase were recorded in pregnant dam serum and tissues. SGOT, SGPT, total bilirubin, urea, uric acid, and creatinine were elevated in ethion groups indicating liver and kidney toxicity. Histology of uterus revealed myometrial degeneration and mucosal gland atrophy in uterus of pregnant dams and degenerative changes in placenta. It showed histological alterations in liver, kidney, and lungs. There was reduction in the foetal body weights and placental weights, and degenerative changes in the foetal liver and kidney. Gross evaluation of foetuses showed subcutaneous hematoma. Skeletal evaluation showed partial ossification of skull bones, costal separation, and agenesis of tail vertebrae, sternebrae, metacarpals and metatarsals. The findings reveal that prenatal exposure to ethion caused maternal and foetal toxicity in rats.

TRPV4 channel activation leads to endothelium-dependent relaxation mediated by nitric oxide and endothelium-derived hyperpolarizing factor in rat pulmonary artery.

The purpose of the present study was to characterize TRPV4 channels in the rat pulmonary artery and examine their role in endothelium-dependent relaxation. Tension, Real-Time polymerase chain reaction (Real-Time PCR) and Western blot experiments were conducted on left and right branches of the main pulmonary artery from male Wistar rats. TRPV4 channel agonist GSK1016790A (GSK) caused concentration-related robust relaxation (Emax 88.6±5.5%; pD2 8.7±0.2) of the endothelium-intact pulmonary artery. Endothelium-denudation nearly abolished the relaxation (Emax 5.6±1.3%) to GSK. TRPV4 channel selective antagonist HC067047 significantly attenuated GSK-induced relaxation (Emax 56.2±6.6% vs. control Emax 87.9±3.3%) in endothelium-intact vessels, but had no effect on either ACh-induced endothelium-dependent or SNP-induced endothelium-independent relaxations. GSK-induced relaxations were markedly inhibited either in the presence of NO synthase inhibitor L-NAME (Emax 8.5±2.7%) or sGC inhibitor ODQ (Emax 28.1±5.9%). A significant portion (Emax 30.2±4.4%) of endothelium-dependent relaxation still persisted in the combined presence of L-NAME and cyclooxygenase inhibitor indomethacin. This EDHF-mediated relaxation was sensitive to inhibition by 60mM K(+) depolarizing solution or K(+) channel blockers apamin (SKCa; KCa2.3) and TRAM-34 (IKCa; KCa3.1). GSK (10(-10)-10(-7)M) caused either modest decrease or increase in the basal tone of endothelium-intact or denuded rings, respectively. We found a greater abundance (>1.5 fold) of TRPV4 mRNA and protein expressions in endothelium-intact vs. denuded vessels, suggesting the presence of this channel in pulmonary endothelial and smooth muscle cells as well. The present study demonstrated that NO and EDHF significantly contributed to TRPV4 channel-mediated endothelium-dependent relaxation of the rat pulmonary artery.

Pravastatin attenuates isoprenaline induced cardiac fibrosis in a mouse model.

We investigated the effects of pravastatin (PRAVA) on isoprenaline (ISP) induced cardiac fibrosis using four groups of mice: untreated control, PRAVA, ISP, ISP + PRAVA groups. ISP, 20 mg/kg, was administered subcutaneously daily for 14 days. PRAVA, 20 mg/kg, was administered orally daily for 14 days. Mice were sacrificed on day15 and heart and blood samples were collected to investigate cardiac injury markers. The mean body weight for the ISP group on day 15 was decreased significantly compared to day 0; PRAVA increased the mean body weight slightly on day 15 of treatment compared to day 0. The heart:body weight ratio was increased in the ISP group compared to the control group, but the ratio was returned to near control ratio in the PRAVA + ISP group. The serum creatine kinase-myocardial band (CK-MB) level was reduced significantly in the PRAVA + ISP group compared to the ISP group. Serum triglyceride level was decreased significantly in ISP + PRAVA group compared to the ISP group. PRAVA administration significantly reduced tissue collagen I and III levels in the ISP + PRAVA group compared to the ISP group. Lipid oxidation was decreased and reduced glutathione activity was increased in the PRAVA + ISP group compared to the ISP group. IL-6, α-SMA, CTGF, TGF-ß and SMAD-3 gene expressions were decreased in the PRAVA + ISP group compared to the ISP group. We found fewer inflammatory cells and less fibrosis in heart tissue in the PRAVA + ISP group compared to the ISP group. PRAVA decreased ISP induced cardiac fibrosis by reducing oxidative stress, collagen deposition and inflammation, as well as by decreasing expression of TGF-ß, SMAD-3 and CTGF genes.

In vivo therapeutic efficacy of Curcuma longa extract loaded ethosomes on wound healing.

Since ancient times, medicinal plants are widely accepted to promote the health and wellness of animals and mankind. The medicinal plant-based therapies have limitations of delayed onset of action, inconsistent absorption, low bioavailability, oxidation, and poor solubility. The encapsulation studies suggested improved efficacy. Therefore, the present study attempts to evaluate the efficacy of Curcuma longa extracts encapsulated in Ethosome on wound healing model compared to crude extract. The Curcuma longa extract swere prepared by cold percolation method and total curcuminoid content was determined by Reverse phase-HPLC. Three Ethosomal suspensions (ETS1, ETS2, and ETS3) were prepared and characterized for particle distribution, morphology, and absorption spectrum by Zetasizer, Scanning Electron Microscopy, and FTIR respectively. The Ethosomal suspension with the highest entrapment efficiency was applied topically at a varying concentrations (0.25, 0.5, and 1 g/cm2) on the surgically created wounds in rats. The efficacy of wound healing was evaluated by clinical observation, macroscopic evaluation of granulation tissue, colour digital image processing, and histology. The methanolic extract of Curcuma longa showed better antibacterial potential than ethanolic and aqueous. The total Curcuminoid content in the Curcuma longa rhizome was 4.03%. The size, PDI, zeta potential, and viscosity of Ethosomal suspension ranged from 34.8 to 371 nm, 0.236-1.178, 15.6-36.8mV, and 0.8460-0.8510, respectively. The ETS3 was found the most optimum combination with the highest entrapment efficiency and the topical application at a dose rate of 0.5 g/cm2 and 1.0 g/cm2 resulted in comparable wound contracture, pain score, histopathological score as compared to control groups.It was concluded that the Curcuma longa encapsulation in Ethosome resulted in improved wound appearance, granulation tissue score, and appearance with a shortened period of wound resolution at the cellular level as compared to crude extract.

Luteolin alleviates vascular dysfunctions in CLP-induced polymicrobial sepsis in mice.

BACKGROUND: Luteolin, a naturally occurring flavonoid, is thought to have health-promoting properties as a part of human diet and has been reported to possess a wide range of pharmacological activities. Therefore, the present study was undertaken to evaluate the effect of luteolin pre-treatment on vascular dysfunctions in sepsis induced by caecal ligation and puncture (CLP) in the mouse model. METHODS: Mice were divided into four groups: sham, luteolin plus sham, CLP, and luteolin plus CLP. Luteolin was administered (0.2 mg/kg body weight) intraperitoneally one hour (h) before CLP surgery in mice. 20 ± 2 h post CLP surgery, the isolated thoracic aorta of mice was assessed for its vascular reactivity to noradrenaline (NA) and acetylcholine (ACh). To explore the underlying mechanism, aortic mRNA expressions of α1D adrenoceptors, eNOS and iNOS were investigated. RESULTS: In mice with CLP-induced sepsis luteolin pre-treatment markedly increased the survival time and attenuated serum lactate level. The CLP group manifested the reduced vascular reactivity to NA and this deficit was restored by luteolin pre-treatment. However, luteolin pre-treatment did not improve α1D adrenoceptors down-regulation observed in septic mice aorta. In the presence of 1400 W, the NA contractile response was significantly restored in CLP mice aortic tissue in comparison with the respective control of septic mice and further enhanced in the presence of luteolin. Luteolin reduced the iNOS mRNA expression and iNOS-derived nitrite production. Pre-treatment with luteolin restored the endothelial dysfunction in septic mice aorta by improving eNOS mRNA expression and enhanced eNOS-derived nitric oxide (NO) production in septic mice aorta and aortic iNOS gene expression and inducible NO production. CONCLUSION: The present study suggests that the vasoplegic state to NA in aorta was restored through the iNOS pathway and endothelial dysfunction was reversed via eNOS and NO production pathway.

PRE-084 ameliorates adenine-induced renal fibrosis in rats.

2-(4-morpholinoethyl)- 1-phenylcyclohexane-1-carboxylate hydrochloride (PRE-084) is a selective sigma 1 receptor agonist. It has been shown that PRE-084 protected various tissues from experimental injury. However, no reports are available on its effect on renal fibrosis. Rat model of adenine-induced chronic kidney disease was chosen to study this. Adenine feeding in rats caused renal dysfunction as shown by increased serum creatinine and reduced creatinine clearance along with increased high molecular weight (HMW) urine protein excretion. Further, adenine feeding induced profibrotic changes in the kidney as reflected by increased expression of alpha-smooth muscle actin (α-SMA), fibroblast specific protein-1 (FSP-1) and matrix metalloproteinase-2 (MMP-2) activity; reduced cytokeratin expression. Further, there was excess deposition of extracellular matrix in the kidney, a striking character of fibrosis. However, administration of PRE-084 to adenine fed rats led to reduction in creatinine and proteinuria parameters partly. This was accompanied by reduced expression of α-SMA, FSP-1 and MMP-2 activity and slight restoration of cytokeratin levels leading to reduced extracellular matrix deposition in the kidney. These data demonstrate that PRE-084 partly ameliorated renal dysfunction and exhibited anti-fibrotic potential in the kidney of adenine fed rats.

Drug repurposing approach to fight COVID-19.

Currently, there are no treatment options available for the deadly contagious disease, coronavirus disease 2019 (COVID-19). Drug repurposing is a process of identifying new uses for approved or investigational drugs and it is considered as a very effective strategy for drug discovery as it involves less time and cost to find a therapeutic agent in comparison to the de novo drug discovery process. The present review will focus on the repurposing efficacy of the currently used drugs against COVID-19 and their mechanisms of action, pharmacokinetics, dosing, safety, and their future perspective. Relevant articles with experimental studies conducted in-silico, in-vitro, in-vivo, clinical trials in humans, case reports, and news archives were selected for the review. Number of drugs such as remdesivir, favipiravir, ribavirin, lopinavir, ritonavir, darunavir, arbidol, chloroquine, hydroxychloroquine, tocilizumab and interferons have shown inhibitory effects against the SARS-CoV2 in-vitro as well as in clinical conditions. These drugs either act through virus-related targets such as RNA genome, polypeptide packing and uptake pathways or target host-related pathways involving angiotensin-converting enzyme-2 (ACE2) receptors and inflammatory pathways. Using the basic knowledge of viral pathogenesis and pharmacodynamics of drugs as well as using computational tools, many drugs are currently in pipeline to be repurposed. In the current scenario, repositioning of the drugs could be considered the new avenue for the treatment of COVID-19.