Dose-dependent Fashion Research Articles

Dose-Dependent Effect of Melatonin on BAT Thermogenesis in Zücker Diabetic Fatty Rat: Future Clinical Implications for Obesity.

Experimental data have revealed that melatonin at high doses reduced obesity and improved metabolic outcomes in experimental models of obesity, mainly by enhancing brown adipose tissue (BAT) thermogenesis. A potential dose-response relationship has yet to be performed to translate these promising findings into potential clinical therapy. This study aimed to assess the effects of different doses of melatonin on interscapular BAT (iBAT) thermogenic capacity in Zücker diabetic fatty (ZDF) rats. At 6 wk of age, male ZDF rats were divided into four groups (n = 4 per group): control and those treated with different doses of melatonin (0.1, 1, and 10 mg/kg of body weight) in their drinking water for 6 wk. Body weight (BW) was significantly decreased at doses of 1 and 10 mg/kg of melatonin, but not at 0.1 mg/kg compared with the control, with a similar rate of BW decrease being reached at the dose of 1 mg/kg (by ~11%) and 10 mg/kg (by ~12%). This effect was associated with a dose-dependent increase in the thermal response to the baseline condition or acute cold challenge in the interscapular area measurable by infrared thermography, with the highest thermal response being recorded at the 10 mg/kg dose. Upon histology, melatonin treatment markedly restored the typical brownish appearance of the tissue and promoted a shift in size distribution toward smaller adipocytes in a dose-dependent fashion, with the most pronounced brownish phenotype being observed at 10 mg/kg of melatonin. As a hallmark of thermogenesis, the protein level of uncoupled protein 1 (UCP1) from immunofluorescence and Western blot analysis increased significantly and dose-dependently at all three doses of melatonin, reaching the highest level at the dose of 10 mg/kg. Likewise, all three doses of melatonin modulated iBAT mitochondrial dynamics by increasing protein expression of the optic atrophy protein type 1 (OPA1) fusion marker and decreasing that of the dynamin-related protein1 (DRP1) fission marker, again dose-dependently, with the highest and lowest expression levels, respectively, being reached at the 10 mg/kg dose. These findings highlight for the first time the relevance of the dose-dependency of melatonin toward BW control and BAT thermogenic activation, which may have potential therapeutic implications for the treatment of obesity. To clinically apply the potential therapeutic of melatonin for obesity, we consider that the effective animal doses that should be extrapolated to obese individuals may be within the dose range of 1 to 10 mg/kg.

Sulforaphane Regulates eNOS Activation and NO Production via Src-Mediated PI3K/Akt Signaling in Human Endothelial EA.hy926 Cells

Sulforaphane (SFN) is a naturally occurring isothiocyanate that is abundant in many cruciferous vegetables, such as broccoli and cauliflower, and it has been observed to exert numerous biological activities. In the present study, we investigate the effect of SFN on eNOS, a key regulatory enzyme of vascular homeostasis and underlying intracellular pathways, in human endothelial EA.hy926 cells. The results indicate that SFN treatment significantly increases NO production and eNOS phosphorylation in a time- and dose-dependent fashion and also augments Akt phosphorylation in a time- and dose-dependent manner. Meanwhile, pretreatment with LY294002 (a specific PI3K inhibitor) suppresses the phosphorylation of eNOS and NO production. Furthermore, SFN time- and dose-dependently induces the phosphorylation of Src kinase, a further upstream regulator of PI3K, while PP2 pretreatment (a specific Src inhibitor) eliminates the increase in phosphorylated Akt, eNOS and the production of NO derived from eNOS. Overall, the present study uncovers a novel effect of SFN to stimulate eNOS activity in EA.hy926 cells by regulating NO bioavailability. These findings provide clear evidence that SFN regulates eNOS activity and NO bioavailability, suggesting a promising therapeutic candidate to prevent endothelial dysfunction, atherosclerosis and other cardiovascular diseases.

Design, Synthesis, and Evaluation of New Mesenchymal-Epithelial Transition Factor (c-Met) Kinase Inhibitors with Dual Chiral Centers.

A series of tepotinib derivatives with two chiral centers was designed, synthesized, and evaluated as anticancer agents. The optimal compound (R, S)-12a strongly exhibited antiproliferative activity against MHCC97H cell lines with an IC50 value of 0.002 μM, compared to tepotinib (IC50 = 0.013 μM). Mechanistic studies revealed that compound (R, S)-12a significantly inhibited c-Met activation, as well as the downstream AKT signaling pathway, and suppressed wound closure. Moreover, compound (R, S)-12a induced cellular apoptosis and cell cycle arrest at the G1 phase in a dose-dependent fashion.

Phytochemical screening and antioxidant activity of Ipomoea hederifolia stems: A potential medicinal plant

Abstract. Hossain MM, Uddin MS, Baral PK, Ferdus M, Bhowmik S. 2022. Phytochemical screening and antioxidant activity of Ipomoea hederifolia stems: A potential medicinal plant. Asian J Nat Prod Biochem 20: 41-47. Ipomoea hederifolia L., a plant of Convolvulaceae family, popularly known as morning glory, possesses numerous medicinal values. The present study aimed to explore the antioxidant activity and bioactive compounds of I. hederifolia stems (IHS). The IHS was soaked in methanol for 21 days. The filtrate was concentrated using rotary evaporator after filtration to obtain IHS extract which is subjected to phytochemical screening using various tests. Antioxidant activity was determined using 1,1-Diphenyl-2-picrylhydrazyl (DPPH) and reducing power activity (RPA) methods. The phytochemical screenings revealed that IHS extract possesses carbohydrates, tannins, flavonoids, phenols, saponin, alkaloids, steroids, anthraquinones, and cardiac glycosides. The antioxidant activity of IHS extract in DPPH model was moderate (IC50 :174.08 µg/ml) in comparison with standards (IC50: 102.28 µg/mL AA; IC50: 88.52 µg/mL for BHT). It indicates that IHS exerts free radical scavenging power in dose dependent fashion. The RPA model also produced moderate antioxidant potential (EC50: 279.58 µg/mL for IHS; EC50: 23.121 µg/mL for AA; EC50: 50.84 µg/mL for BHT) depending on increasing order of dose. Based on the findings of this investigation, we can conclude that IHS extract possesses various bioactive compounds and moderate antioxidant potentials which may be path to discovery of traditional medicines and remedies for many critical diseases.

Coptisine Reduces the Progression of Cervical Cancer Through Induction of Autophagy

The regulatory role of coptisine on autophagy in cervical cancer cells (in HcerEpic, HeLa, and SiHa) was examined by assessment of cell viability, colony formation, and apoptosis. Coptisine, in a dose-dependent fashion, significantly inhibited cervical cancer cell viability as measured by cell proliferation, transwell, and TUNEL assays. Coptisine inhibited the levels of p62 and Bcl-2, whilst it promoted the accumulation of Atg5, LC3Ⅱ/LC3Ⅰ and Bax. These data taken together suggest that coptisine inhibits the progression of cervical cancer cells by enhancing autophagy through suppression of the PI3K/AKT/mTOR pathway. This represents a theoretical foundation for developing novel cervical cancer treatments.

Novel identification and modulation of the mechanosensitive Piezo1 channel in human myometrium.

Approximately 10% of US births deliver preterm before 37weeks of completed gestation. Premature infants are at risk for life-long debilitating morbidities and death, and spontaneous preterm labour explains 50% of preterm births. In all cases existing treatments are ineffective, and none are FDA approved. The mechanisms that initiate preterm labour are not well understood but may result from dysfunctional regulation of quiescence mechanisms. Human pregnancy is accompanied by large increases in blood flow, and the uterus must enlarge by orders of magnitude to accommodate the growing fetus. This mechanical strain suggests that stretch-activated channels may constitute a mechanism to explain gestational quiescence. Here we identify for the first time that Piezo1, a mechanosensitive cation channel, is present in the uterine smooth muscle and microvascular endothelium of pregnant myometrium. Piezo is downregulated during preterm labour, and stimulation of myometrial Piezo1 in an organ bath with the agonist Yoda1 relaxes the tissue in a dose-dependent fashion. Further, stimulation of Piezo1 while inhibiting protein kinase A, AKT, or endothelial nitric oxide synthase mutes the negative inotropic effects of Piezo1 activation, intimating that actions on the myocyte and endothelial nitric oxide signalling contribute to Piezo1-mediated contractile dynamics. Taken together, these data highlight the importance of stretch-activated channels in pregnancy maintenance and parturition, and identify Piezo1 as a tocolytic target of interest. KEY POINTS: Spontaneous preterm labour is a serious obstetric dilemma without a known cause or effective treatments. Piezo1 is a stretch-activated channel important to muscle contractile dynamics. Piezo1 is present in the myometrium and is dysregulated in women who experience preterm labour. Activation of Piezo1 by the agonist Yoda1 relaxes the myometrium in a dose-dependent fashion, indicating that Piezo1modulation may have therapeutic benefits to treat preterm labour.

Abstract P1015: Construction Of A Pro-cardiogenic Extracellular Matrix: How Stem-cell Derived Epicardium Enhances Cardiac Regeneration Through Fibronectin

Background: Human embryonic stem cell-derived epicardium (hESC-Epis) has been shown to improve the function of hESC-derived cardiomyocytes (hESC-CM) in vitro and post-transplantation into animal myocardial infarction models. However, the underlying mechanisms mediating these effects remain poorly characterised. Bulk RNA sequencing of hESC-Epis suggests a role for genes related to extracellular matrix remodeling in mediating these effects, with fibronectin (FN1) being highly implicated. Study Objective: To examine the role of FN1 in hESC-CM/Epi crosstalk through gain and loss of function studies. Methods: A tetracycline inducible FN1 knockdown system was used on hESC-CMs and Epis within the setting of 3D-engineered heart tissue. Changes in contractility and calcium handling were measured. Recombinant human plasma FN1 (rhFN) was then used to determine the effect of FN1 supplementation on hESC-CM function. Results: FN1 knockdown in hESC-Epis resulted in significant abrogation of the beneficial effects on hESC-CM force generation and calcium handling. Knockdown of FN1 in hESC-CMs however significantly attenuated contractility of the hESC-CMs but had no effects on calcium handling. When rhFN was added to hESC-CMs in increasing doses, hESC-CM contractility was improved in a dose dependent fashion to the same extent as seen with hESC-Epis co-cultures. However, rhFN supplementation did not result in significant improvements in calcium handling. Conclusions: hESC-CM and Epi crosstalk improving hESC-CM function may rely heavily on the presence of FN1. FN1 secreted by both cell types are shown to be of importance in this crosstalk with artificial supplementation of rhFN being able to recapitulate some beneficial effects of hESC-Epis on hESC-CMs. Further studies are required to determine the role of specific isoforms of secreted FN1 on hESC-CM maturity and what mechanism underlies these effects. This opens up the possibility of improving cell-based therapy for heart failure post-myocardial infarction using recombinant FN1 supplementation.

Ipatasertib, an oral AKT inhibitor, in combination with paclitaxel exhibits anti-proliferative and anti-tumorigenic effects in preclinical studies for endometrioid endometrial cancer: Endometrial cancer molecularly targeted therapy consortium (227)

<b>Objectives:</b> Ipatasertib (IPAT) is an orally administered selective protein kinase B (AKT) inhibitor that has demonstrated clinical activity in triple-negative breast and metastatic prostate cancer. The PI3K/ AKT pathway is altered in the vast majority of endometrioid endometrial cancers (ECs). A recent clinical trial demonstrated that IPAT combined with paclitaxel showed a significant prolonged overall survival in breast cancer patients. Thus, we aimed to evaluate the antitumorigenic effects of IPAT in combination with paclitaxel in EC cell lines and an EC mouse model. <b>Methods:</b> The human endometrioid EC cell lines Ishikawa <i>(PTEN</i> mutant), and HEC-1A (<i>PTEN</i> wild type, <i>PIK3CA</i> mutant, <i>KRAS</i> mutant) were exposed to varying concentrations and durations of IPAT (Genentech) and paclitaxel. Cell proliferation was assessed by MTT assay. To assess the synergy of IPAT and paclitaxel, the combination index (CI) was calculated by the method of Chou and Talalay. Apoptosis was assessed by cleaved caspase-3 assay. Western immunoblotting determined the effects of IPAT and paclitaxel on apoptosis (MCL, BCL-xl), cellular stress (ATF, PERK, Bip, PDI, IER), and DNA damage (RAD50). LKB1^fl/^flp53^fl/fl mice were used to evaluate the effect of IPAT, paclitaxel, and the combination of these two drugs on EC growth. The mice were treated with placebo, IPAT (50mg/kg daily orally for four weeks), paclitaxel (10 mg/kg IP weekly), or IPAT/paclitaxel starting eight weeks after tumor induction via AdCre injection. <b>Results:</b> IPAT and paclitaxel inhibited cellular proliferation in a dose-dependent fashion in both cell lines after 72 hours of treatment. The median IC50 for IPAT was 0.13µM for Ishikawa and 4.9µM for HEC-1A, respectively. The median IC50 for paclitaxel was 5.6nM for Ishikawa and 1.9nM for HEC-1A, respectively. Simultaneous exposure of cells to various doses of paclitaxel in combination with IPAT resulted in a significant synergistic antiproliferative effect (CI<1). IPAT and paclitaxel induced the activity of cleaved caspase-3 by 3.5-fold in Ishikawa and 1.12-fold in HEC-1A, while simultaneously increasing expression of the apoptotic proteins MCL-1 and BCL-xl. IPAT and paclitaxel also increased Bip, PERK, ATF4, PDI, and IER protein expression. Treatment of cells with IPAT and paclitaxel decreased phosphorylated-S6 (p-S6) expression and induced the expression of p-AKT and RAD50. Compared to control mice, treatment of LKB1^fl/flp53^fl/fl mice with IPAT reduced tumor weight by 62.1%, and treatment with paclitaxel decreased tumor weight by 73.9% (p<0.05). The combination of IPAT and paclitaxel reduced tumor growth by 78.3% and showed improved efficacy over IPAT alone (p<0.05), but not over Taxol alone. <b>Conclusions:</b> The combination of IPAT and paclitaxel demonstrates synergy in human endometrioid EC cell lines and improved efficacy over IPAT alone in the LKB^1fl/fl/p53^fl/fl mouse model of EC. These results suggest that IPAT with paclitaxel is worthy of further exploration in clinical trials for EC.

Oleic acid, a monounsaturated fatty acid, exhibits antiproliferative and anti-tumorigenic effects in pre-clinical studies for endometrioid endometrial cancer (261)

<b>Objectives:</b> Oleic acid (OA), a monounsaturated fatty acid commonly found in olive oil, has been studied for its potential protective qualities in inflammatory conditions and cancers. In breast cancer cells, OA has been shown to reduce the expression of the oncogene <i>Her-2/ neu</i> and promote apoptosis. The relationship between the intake of olive oil and endometrial cancer risk and progression has been largely unexplored. Thus, we aimed to evaluate the effect of OA on cell proliferation and tumor growth in endometrial cancer cell lines and a transgenic mouse model of endometrial cancer. <b>Methods:</b> The human endometrioid endometrial cancer cell lines, ECC-1 and KLE, were exposed to varying concentrations of OA. Cell proliferation was assessed by MTT assay. Apoptosis was assessed by cleaved caspase-3, -8, and -9 assays. Cell cycle progression was measured by Cellometer. Cellular stress was measured by DCFH-DA assay. Mitochondrial membrane potential was assessed by TMRE assay. Western immunoblotting was performed to determine the effects of OA on BCL-xl, MCL-1, CDK4, CyclinD1, Calnexin, and Bip. The LKB1^fl/flp53^fl/fl mouse model of endometrioid endometrial cancer was used to determine the antitumorigenic activity of OA. The mice were treated with OA (10 mg/kg, oral gavage, daily) versus control for four weeks. <b>Results:</b> OA inhibited proliferation in a dose-dependent manner in both cell lines, with a 60% reduction at a dose of 500 µM in the ECC-1 cell line and 250 µM in the KLE cell line. OA at a dose of 200 µM significantly increased the activity of cleaved caspase-3, 8, and 9 by 2.2, 1.4, and 1.7-fold in the ECC-1 cells (p<0.05), respectively, and 1.4, 1.3, and 1.4-fold in the KLE cells (p<0.05), respectively. OA simultaneously decreased expression of Bcl-xl and MCL-1 in both cell lines. OA inhibited cell cycle progression by G1 phase arrest in both cell lines in a dose-dependent fashion (p=0.02) and decreased expression of the cell cycle-related proteins, CDK4, and cyclin D1. Treatment with OA significantly increased ROS production at doses of 50 and 200 µM (p<0.05) and induced Calnexin and Bip expression in both cell lines. TMRE assay showed decreased mitochondrial membrane potential with OA at doses of 50 and 200 µM (p<0.05). Treatment of LKB1^fl/flp53^fl/fl mice with OA was well-tolerated and reduced tumor weight by 53% compared with control mice (p<0.05). <b>Conclusions:</b> We found that OA, the main monounsaturated fatty acid of olive oil, had antiproliferative and antitumorigenic effects in endometrioid endometrial cancer. These findings provide a preclinical rationale for OA as a potential dietary intervention and therapeutic adjunct in endometrial cancer patients.

FGF8-FGFR1 signaling regulates human GnRH neuron differentiation in a time- and dose-dependent manner.

ABSTRACTFibroblast growth factor 8 (FGF8), acting through the fibroblast growth factor receptor 1 (FGFR1), has an important role in the development of gonadotropin-releasing hormone-expressing neurons (GnRH neurons). We hypothesized that FGF8 regulates differentiation of human GnRH neurons in a time- and dose-dependent manner via FGFR1. To investigate this further, human pluripotent stem cells were differentiated during 10 days of dual-SMAD inhibition into neural progenitor cells, followed either by treatment with FGF8 at different concentrations (25 ng/ml, 50 ng/ml or 100 ng/ml) for 10 days or by treatment with 100 ng/ml FGF8 for different durations (2, 4, 6 or 10 days); cells were then matured through DAPT-induced inhibition of Notch signaling for 5 days into GnRH neurons. FGF8 induced expression of GNRH1 in a dose-dependent fashion and the duration of FGF8 exposure correlated positively with gene expression of GNRH1 (P<0.05, Rs=0.49). However, cells treated with 100 ng/ml FGF8 for 2 days induced the expression of genes, such as FOXG1, ETV5 and SPRY2, and continued FGF8 treatment induced the dynamic expression of several other genes. Moreover, during exposure to FGF8, FGFR1 localized to the cell surface and its specific inhibition with the FGFR1 inhibitor PD166866 reduced expression of GNRH1 (P<0.05). In neurons, FGFR1 also localized to the nucleus. Our results suggest that dose- and time-dependent FGF8 signaling via FGFR1 is indispensable for human GnRH neuron ontogeny. This article has an associated First Person interview with the first author of the paper.

STAT3 is a potential therapeutic target in cervical cancer (257)

<b>Objectives:</b> STAT3 is known to be hyperactivated in most human cancers and is associated with poor prognoses. The STAT3 activation pathway within human papillomavirus (HPV)-positive cervical cancer cells is known to be essential for cervical cancer cell proliferation and survival. However, whether STAT3 activity is elevated in cervical cancer patient tumors remains to be determined. We sought to assess whether activated STAT3 (p-STAT3) was more highly expressed in cervical squamous cell carcinoma tissue microarrays compared to benign adjacent cervix tissue. We also assessed the ability of a small molecule inhibitor of STAT3 that has been used in the clinic to attenuate the STAT3 activation pathway and induce tumor cell death of HPV-associated human squamous cell carcinoma cells <i>in vitro</i>. <b>Methods:</b> Tumor microarrays from 12 patients with cervical squamous cell carcinoma with adjacent, benign cervix tissue were first probed with an anti-p-STAT3 antibody and detected with a fluorophore-conjugated secondary antibody for p-STAT3. Confocal imaging of immunofluorescence was performed, and fluorescence quantification was performed by ZEN 2.3 lite software and plotted in GraphPad Prism 8 software. The CaSki HPV16 infected cervical squamous cell carcinoma cell line was used for <i>in vitro</i> experiments. CaSki cells were treated with BBI608 (a small molecule inhibitor of STAT3) and assessed for changes in the STAT3 activation axis using Western Blot. Cell viability was analyzed using CellTiter-Glo® Luminescent Cell Viability Assay. <b>Results:</b> Quantification of p-STAT3 immunofluorescence from confocal imaging showed significantly higher expression of p-STAT3 in cervical squamous cell carcinoma tissue microarrays compared to adjacent, benign cervix tissue with mean expression levels of 37.7 relative units (RU) vs 29.1 RU, respectively (p=0.006). On Western Blot, BBI608 (3uM and 4uM) was found to attenuate the STAT3 activation axis in a dose-dependent fashion, including a significant decrease in p-JAK1 and p-STAT3 protein expressions. Furthermore, the protein expression of <i>HIF1A</i>, a known downstream STAT3 target gene and associated with poor survival and treatment resistance in cervical cancer, was also decreased by BBI608 (3uM and 4uM) in a similar fashion. On analysis of cell viability, BBI608 was found capable of inducing cell death in CaSki cells with an IC50 of 2.6uM after a 48-hour incubation period. <b>Conclusions:</b> The STAT3 activation pathway is a logical and promising target for therapeutic intervention in cervical cancer. On review of HPV-associated cervical cancer tumor arrays, we found a significantly higher expression of pSTAT3 in cervical tumor cells and tumor-infiltrating lymphocytes compared to adjacent, benign tissue. Furthermore, we found that BBI608 was able to attenuate the STAT3 activation axis and induce cell death in the CaSki cervical squamous cell carcinoma cell line.Fig. 1

SOX transcription factors direct TCF-independent WNT/β-catenin responsive transcription to govern cell fate in human pluripotent stem cells.

WNT/β-catenin signaling controls gene expression across biological contexts from development and stem cell homeostasis to diseases including cancer. How β-catenin is recruited to distinct enhancers to activate context-specific transcription is unclear, given that most WNT/ß-catenin-responsive transcription is thought to be mediated by TCF/LEF transcription factors (TFs). With time-resolved multi-omic analyses, we show that SOX TFs can direct lineage-specific WNT-responsive transcription during the differentiation of human pluripotent stem cells (hPSCs) into definitive endoderm and neuromesodermal progenitors. We demonstrate that SOX17 and SOX2 are required to recruit β-catenin to lineage-specific WNT-responsive enhancers, many of which are not occupied by TCFs. At TCF-independent enhancers, SOX TFs establish a permissive chromatin landscape and recruit a WNT-enhanceosome complex to activate SOX/ß-catenin-dependent transcription. Given that SOX TFs and the WNT pathway are critical for specification of most cell types, these results have broad mechanistic implications for the specificity of WNT responses across developmental and disease contexts.

A conserved annexin A6-mediated membrane repair mechanism in muscle, heart, and nerve.

Membrane instability and disruption underlie myriad acute and chronic disorders. Anxa6 encodes the membrane-associated protein annexin A6 and was identified as a genetic modifier of muscle repair and muscular dystrophy. To evaluate annexin A6’s role in membrane repair in vivo, we inserted sequences encoding green fluorescent protein (GFP) into the last coding exon of Anxa6. Heterozygous Anxa6gfp mice expressed a normal pattern of annexin A6 with reduced annexin A6GFP mRNA and protein. High-resolution imaging of wounded muscle fibers showed annexin A6GFP rapidly formed a repair cap at the site of injury. Injured cardiomyocytes and neurons also displayed repair caps after wounding, highlighting annexin A6–mediated repair caps as a feature in multiple cell types. Using surface plasmon resonance, we showed recombinant annexin A6 bound phosphatidylserine-containing lipids in a Ca2+- and dose-dependent fashion with appreciable binding at approximately 50 μM Ca2+. Exogenously added recombinant annexin A6 localized to repair caps and improved muscle membrane repair capacity in a dose-dependent fashion without disrupting endogenous annexin A6 localization, indicating annexin A6 promotes repair from both intracellular and extracellular compartments. Thus, annexin A6 orchestrates repair in multiple cell types, and recombinant annexin A6 may be useful in additional chronic disorders beyond skeletal muscle myopathies.

In vitro and in vivo efficacies of Dectin-1-Fc(IgG)(s) fusion proteins against invasive fungal infections.

Mycoses have increased worldwide, and new efficient therapeutics are needed. Passive immunizations targeting universally the fungal cell would allow early interventions without the species-level diagnosis. Lectins with affinity to carbohydrates could be used to engineer 'antibody-like' strategies.

Discovery and antiviral profile of new sulfamoylbenzamide derivatives as HBV capsid assembly modulators

Chronic hepatitis B (CHB) is a major worldwide public health problem and novel anti-HBV therapies preventing liver disease progression to cirrhosis and hepatocellular carcinoma are urgently needed. Over the last several years, capsid assembly modulators (CAM) have emerged as clinically effective anti-HBV agents which can inhibit HBV replication in CHB patients. As part of a drug discovery program aimed at obtaining novel CAM endowed with high in vitro and in vivo antiviral activity, we identified a novel series of sulfamoylbenzamide (SBA) derivatives. Compound 10, one of the most in vitro potent SBA-derived CAM discovered to date, showed excellent pharmacokinetics in mice suitable for oral dosing. When studied in a transgenic mouse model of hepatic HBV replication, it was considerably more potent than NVR 3–778, the first sulfamoylbenzamide (SBA) CAM that entered clinical trials for CHB, at reducing viral replication in a dose-dependent fashion. We present herein the discovery process, the SAR analysis and the pre-clinical profile of this novel SBA CAM.

Repression of inflammatory pathways with Boswellia for alleviation of liver injury after renal ischemia reperfusion

AimAcute kidney injury (AKI) is a sudden incident that is linked with a high lethality rate commonly due to distant organ injury. This study aims to explore the role of standardized Boswellia serrata (containing 35 % boswellic acid) in attenuating kidney and liver damage in a model of rats with renal insult. Main methodsSprague-Dawley rats, exposed to renal injury via ischemia-reperfusion model, were administered a daily regimen of 1000 or 2000 mg/kg Boswellia for seven days then rats were sacrificed on day eight. Alanine aminotransferase, aspartate aminotransferase, serum creatinine and blood urea nitrogen, were assayed. TLR9, oxidative stress markers; namely MDA and GSH, inflammatory cytokines; namely, IL-6, IL-1β, and TNF-α, as well as NF-κB were also measured. Key findingsRenal ischemia-reperfusion injury (IRI) impaired renal and liver function significantly, but Boswellia attenuated this impairment in a dose-dependent fashion. Histopathological assessment of kidney and liver confirmed that Boswellia decreased damage severity. A marked increase in TLR9, NF-κB, IL-6, IL-1β, TNF-α, and MDA besides decreased GSH levels were observed in the kidney and liver after renal IRI. Boswellia attenuated increases in TLR9, NF-κB, IL-1β, TNF-α, and IL-6 and boosted antioxidant defences via decreasing MDA and increasing GSH in kidney and liver. Anti-inflammatory and antioxidant effects of Boswellia were mostly comparable to those of silymarin. SignificanceWe conclude that the anti-inflammatory and antioxidant effects of Boswellia could be beneficial in ameliorating kidney and liver damage after AKI and that TLR9 might be the connection that signals liver injury in response to renal damage.

Procyanidin B2 Alleviates Heat-Induced Oxidative Stress through the Nrf2 Pathway in Bovine Mammary Epithelial Cells.

The objective of this study was to investigate the protective effects and potential molecular mechanisms of procyanidin B2 (PB2) in MAC-T (mammary alveolar cells-large T antigen) cells during heat stress (HS). The MAC-T cells were divided into three treatment groups: control (37 °C), HS (42 °C), and PB2 + HS (42 °C). Compared with MAC-T cells that were consistently cultured at 37 °C, acute HS treatment remarkably decreased cell viability, reduced activities of catalase (CAT), superoxide dismutase (SOD), and total antioxidant capacity (T-AOC), and elevated intracellular levels of malondialdehyde (MDA) and reactive oxygen species (ROS). Additionally, nuclear factor erythroid 2-related factor 2 (Nrf2) was activated and translocated to the nucleus, in accompaniment with upregulation of Nrf2, heme oxygenase 1 (HO-1), thioredoxin reductase 1 (Txnrd1), and heat shock protein 70 (HSP70). In parallel, both mRNA transcript and actual protein secretion of pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), were increased by heat stress. Pretreatment of MAC-T cells with 0~25 μM PB2 alleviated the decline of cell viability by HS in a dose-dependent fashion and protected cells against HS-induced oxidative stress, as evidenced by significantly improved CAT, SOD, and T-AOC activity, as well as with decreased MDA and ROS generation. Furthermore, PB2 further activated the Nrf2 signaling pathway and reversed the inflammatory response induced by HS. Silencing of Nrf2 by si-Nrf2 transfection not only exacerbated HS-induced cell death and provoked oxidative stress and the inflammatory response, but also greatly abolished the cytoprotective effects under HS of PB2. In summary, PB2 protected MAC-T cells against HS-induced cell death, oxidative stress, and inflammatory response, partially by operating at the Nrf2 signal pathway.

Sphingosine 1-Phoshpate Receptors are Located in Synapses and Control Spontaneous Activity of Mouse Neurons in Culture

Sphingosine-1-phosphate (S1P) is best known for its roles as vascular and immune regulator. Besides, it is also present in the central nervous system (CNS) where it can act as neuromodulator via five S1P receptors (S1PRs), and thus control neurotransmitter release. The distribution of S1PRs in the active zone and postsynaptic density of CNS synapses remains unknown. In the current study, we investigated the localization of S1PR1-5 in synapses of the mouse cortex. Cortical nerve terminals purified in a sucrose gradient were endowed with all five S1PRs. Further subcellular fractionation of cortical nerve terminals revealed S1PR2 and S1PR4 immunoreactivity in the active zone of presynaptic nerve terminals. Interestingly, only S1PR2 and S1PR3 immunoreactivity was found in the postsynaptic density. All receptors were present outside the active zone of nerve terminals. Neurons in the mouse cortex and primary neurons in culture showed immunoreactivity against all five S1PRs, and Ca2+ imaging revealed that S1P inhibits spontaneous neuronal activity in a dose-dependent fashion. When testing selective agonists for each of the receptors, we found that only S1PR1, S1PR2 and S1PR4 control spontaneous neuronal activity. We conclude that S1PR2 and S1PR4 are located in the active zone of nerve terminals and inhibit neuronal activity. Future studies need to test whether these receptors modulate stimulation-induced neurotransmitter release.

&lt;em&gt;In vitro&lt;/em&gt; anti-lipid peroxidation and anti-&lt;em&gt;Candidal &lt;/em&gt;potentials of different betel leaf varieties

Betel leaf (Piper betel L.) is an important plant being widely consumed worldwide as well as being used in traditional medicine. The present study aims to evaluate the anti-lipid peroxidation and anti-Candida potentials of different varieties of betel leaf extracts. Three widely consumed betel leaf varieties of India (Kolkata Variety, Varanasi Variety, and Mysore Variety) were used to evaluate anti-lipid peroxidation, and anti-Candida potentials in different extracts (ethyl acetate, acetone, and methanol). Total Phenolic Content (TPC) and Total Flavonoid Content (TFC) were found to be higher in the ethyl acetate extract of all the varieties and hence selected for medicinal potential evaluation. The Mysore variety exhibited higher antioxidant and free radical scavenging properties IC50 value 179.90 ± 2.98 μg/mL and 319.52 ± 4.00 μg/mL respectively. The Varanasi variety with the IC50 value of 51.54 ± 1.81 μg/mL has slightly high anti-lipid peroxidation potentials over the Mysore variety (IC50 value 61.67 ± 2.92 μg/mL) over the standard butylated hydroxytoluene (BHT) with an IC50 value of 276.33 ± 15.20 μg/mL. The oral lotion disinfectant formulation showed anti-Candida activity in a dose-dependent fashion and the activity was higher than that of the standard drug fluconazole with the inhibition zone of 15mm over fluconazole having zone of inhibition of 12mm. The present study confirmed that the antioxidant and anti-lipid peroxidation potentials of the ethyl acetate extract of the Mysore and Varanasi variety respectively. The study also confirmed the anti-Candida activity of the ethyl acetate extract of betel leaf.

Dose Dependent Effects of Breynia cernua Against the Paraquat Induced Parkinsonism like Symptoms in Animals' Model: In Vitro, In Vivo and Mechanistic Studies.

The aims and objectives of the study were to evaluate the antiParkinson’s (PD) potential of B cernua (BCE). B cernua (Poir.) Müll. Arg. (B cernua) is a member of the Phyllanthaceae family. HPLC revealed the presence of various phytochemicals. Study was conducted for 40 days. After PD induction by paraquat behavioural studies were carried out. Biochemical parameters such as DPPH, NO-scavenging, Ferrous reducing power, MDA, GSH, CAT, SOD, acetylcholinesterase (AChE), neurotransmitter estimation and TNF-α and IL-6 levels were determined. DPPH, NO-scavenging and Ferrous reducing power assays showed 78.02%, 48.05% and 71.45% inhibitions, respectively. There was significant improvement in motor functions and coordination in a dose-dependent manner (50 < 250 < 500 mg/kg) in PD rat model. Biochemical markers; SOD, CAT, GPx and GSH showed significant restoration (P < .001) while MDA showed significant decrease (P < .05). The AChE level was significantly reduced (P < .05) at 500 mg/kg while neurotransmitters were significantly improved (P < .001) in a dose-dependent fashion. The ELISA results showed significant (P < .001) down-regulation of IL-6 and TNF-α level. In conclusion, it is suggested that BCE has the potential to reduce the symptoms of PD.