Effects of long-term anti-ischemic drug treatment on Na,K-ATPase isoforms in cardiomyopathic hamsters

We investigated the effects of long-term anti-ischemic therapy with trimetazidine on Na,K-ATPase (NKA) activity and protein expression in cardiomyopathy. NKA isoforms in membrane fractions from cardiomyopathic hamsters of the BIO 14.6 strain were studied and compared with those from healthy Syrian golden hamsters (F1B). Trimetazidine was orally administered to a subset of cardiomyopathic hamsters in the early stage of active disease (30 days) until the congestive stage (350 days). In the congestive stage of cardiac failure, the cardiomyopathic hamsters displayed altered NKA activity (-55 % vs. F1B; p<0.01), which was related to a specific decrease in abundance of the membrane NKA ?1 isoform (-27 % vs. F1B). Trimetazidine partially prevented the cardiomyopathy-induced changes in NKA activity (+38 %) and ?1 membrane expression (+ 66 %) without inducing changes in the expression of the ?2 isoform or 1 isoform of NKA. Cardiac hypertrophy and remodeling were reduced after trimetazidine treatment. Additionally, the abundance of NKA ?1 in membranes was negatively correlated with the ventricular weight/body weight ratio (an index of cardiac hypertrophy) (r2 = 0.99; p<0.0015). These findings suggest that some of the cardioprotective effect of trimetazidine during long-term cardiomyopathy may be achieved via regulation of cardiac remodeling and selective modulation cardiac NKA isoforms.

Phloretin Ameliorates Testosterone-Induced Benign Prostatic Hyperplasia in Rats by Regulating the Inflammatory Response, Oxidative Stress and Apoptosis

The inflammatory process is proposed to be one of the factors to benign prostatic enlargement (BPH), and this is the first study examining the anti-inflammatory ability of phloretin in treating rats with testosterone-induced BPH. BPH would be induced by testosterone (10 mg/kg/day testosterone subcutaneously for 28 days), and the other groups of rats were treated with phloretin 50 mg/kg/day or 100 mg/kg/day orally (phr50 or phr100 group) after induction. Prostate weight and prostate weight to body weight ratio were significantly reduced in the Phr100 group. Reduced dihydrotestosterone without interfering with 5α-reductase was observed in the phr100 group. In inflammatory proteins, reduced IL-6, IL-8, IL-17, NF-κB, and COX-2 were seen in the phr100 group. In reactive oxygen species, malondialdehyde was reduced, and superoxide dismutase and glutathione peroxidase were elevated in the phr100 group. In apoptotic assessment, elevated cleaved caspase-3 was observed in rats of the phr100 group. Enhanced pro-apoptotic Bax and reduced anti-apoptotic Bc1-2 could be seen in the phr100 group. In histological stains, markedly decreased glandular hyperplasia and proliferative cell nuclear antigen were observed with reduced expression in the phr100 group. Meanwhile, positive cells of terminal deoxynucleotidyl transferase dUTP nick end labeling were increased in the phr100 group. In conclusion, the treatment of phloretin 100 mg/kg/day could ameliorate testosterone-induced BPH.

Nigella Sativa Prevented Parkinson's-Like Motor Functions Impairment, Dopamine Depletion and Neuronal Degeneration in the Striatum of Mptp-Induced Balb/C Mice

Background Parkinsonism is a neurological disease characterised by dopaminergic neuron degeneration in the substantial nigra and dopamine deficiency in the brain, with motor and psycho-cognitive implications, while limitations masked the efficacy of the available drugs, thus the need to find alternatives with less side effects are essential. Nigella sativa is a multi-potent plant with therapeutic potentials in the brain and other body organs. This study investigated the effects of Nigella sativa oil (NSO) on the cognitive and other Parkinsonism endophenotypes elicited by MPTP in the BALB/c strain mice. Materials and Methods Body weights, brain-body ratios, recognition memory (through novel object recognition test), as well as fronto-cortical, striatal and cerebellar dopamine and neuronal density were assayed in thirty-two (32) male BALB/c mice (18 g − 25 g). They were randomized into four groups exposed to; normal feed, 18 mg/kg MPTP i.p, 1 ml/kgbw NSO p.o., and NSO + MPTP respectively, for 5 consecutive days. Behaviours were analysed 24 hours after the last exposure, subsequently euthanized, the brains removed and processed for biomarkers analysis and histochemistry. Results Parkinsonism-like traits such as mild tremor, down-regulation of striatal and fronto-cortical dopamine and neurons were recorded in the BALB/c mice administered with MPTP only. However, significant increase (p < 0.05) in appetite, body weight, brain-body weight ratio, and recognition memory was also recorded in the MPTP-administered mice, though Nigella sativa was significantly prophylactic against the negative Parkinsonic features, and ‘moderative’ of the up-regulations induced by MPTP. Conclusion While this suggests selective MPTP sensitivity and resistance in BALB/c strains, this study recommends the investigation of possible (though ironic) beneficial potentials of MPTP.

Lipoic Acid Exacerbates Oxidative Stress and Lipid Accumulation in the Liver of Wistar Rats Fed a Hypercaloric Choline-Deficient Diet

Non-alcoholic fatty liver disease (NAFLD) is currently estimated as the most prevalent chronic liver disease in all age groups. An increasing body of evidence obtained in experimental and clinical data indicates that oxidative stress is the most important pathogenic factor in the development of NAFLD. The study aimed to investigate the impact of α-lipoic acid (LA), widely used as an antioxidant, on the effects of a hypercaloric choline-deficient diet. Male Wistar rats were divided into three groups: control diet (C); hypercaloric choline-deficient diet (HCCD), and hypercaloric choline-deficient diet with α-lipoic acid (HCCD+LA). Supplementation of HCCD with LA for eight weeks led to a decrease in visceral adipose tissue/body weight ratio, the activity of liver glutathione peroxidase and paraoxonase-1, plasma, and liver total antioxidant activity, as well as an increase in liver/body weight ratio, liver total lipid and triglyceride content, and liver transaminase activities compared to the HCCD group without LA. In conclusion, our study shows that α-lipoic acid detains obesity development but exacerbates the severity of diet-induced oxidative stress and lipid accumulation in the liver of male Wistar rats fed a hypercaloric choline-deficient diet.

Bawei Chenxiang Wan Ameliorates Cardiac Hypertrophy by Activating AMPK/PPAR-α Signaling Pathway Improving Energy Metabolism

Bawei Chenxiang Wan (BCW), a well-known traditional Chinese Tibetan medicine formula, is effective for the treatment of acute and chronic cardiovascular diseases. In the present study, we investigated the effect of BCW in cardiac hypertrophy and underlying mechanisms. The dose of 0.2, 0.4, and 0.8 g/kg BCW treated cardiac hypertrophy in SD rat model induced by isoprenaline (ISO). Our results showed that BCW (0.4 g/kg) could repress cardiac hypertrophy, indicated by macro morphology, heart weight to body weight ratio (HW/BW), left ventricle heart weight to body weight ratio (LVW/BW), hypertrophy markers, heart function, pathological structure, cross-sectional area (CSA) of myocardial cells, and the myocardial enzymes. Furthermore, we declared the mechanism of BCW anti-hypertrophy effect was associated with activating adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK)/peroxisome proliferator–activated receptor-α (PPAR-α) signals, which regulate carnitine palmitoyltransferase1β (CPT-1β) and glucose transport-4 (GLUT-4) to ameliorate glycolipid metabolism. Moreover, BCW also elevated mitochondrial DNA-encoded genes of NADH dehydrogenase subunit 1(ND1), cytochrome b (Cytb), and mitochondrially encoded cytochrome coxidase I (mt-co1) expression, which was associated with mitochondria function and oxidative phosphorylation. Subsequently, knocking down AMPK by siRNA significantly can reverse the anti-hypertrophy effect of BCW indicated by hypertrophy markers and cell surface of cardiomyocytes. In conclusion, BCW prevents ISO-induced cardiomyocyte hypertrophy by activating AMPK/PPAR-α to alleviate the disturbance in energy metabolism. Therefore, BCW can be used as an alternative drug for the treatment of cardiac hypertrophy.

Interleukin-17 aggravates right ventricular remodeling via activating STAT3 under both normoxia and hypoxia

Objective Proinflammatory cytokine interleukin 17 (IL-17) is involved in ventricular remodeling, mainly of the left ventricle. This study was designed to explore the role of IL-17 played in the pathogenesis of right ventricular hypertrophy (RVH), aiming to provide a novel treatment target or diagnostic biomarker options for improving the care of RVH patients. Methods C57BL/6 mice were maintained in 10% O2 chamber or room air for four weeks. Right ventricular hypertrophy index (RVHI), RV/body weight ratio, pulmonary arteriolar remodeling determined by percent media thickness (%MT), and the cardiomyocyte diameter of RV were evaluated. Mice were treated with exogenous recombinant mouse IL-17 (rmIL-17, 1 μg per dose twice a week) for four weeks. H9c2 cardiomyocytes were cultured and treated with IL-17 (10 ng/mL) and STAT3 inhibitor (10 ng/mL) either under normoxia (21% O2, 5% CO2, 74% N2) or under hypoxia (3% O2, 5% CO2, 92% N2). Cardiomyocyte viability was assessed by Cell counting kit 8 (CCK-8) assay. The mRNA level was detected by RT-PCR, where as the protein expression was measured by Western blot, immunohistochemistry, and immunofluorescent analyses. Results In vivo experiments showed that IL-17 did not affect the pulmonary artery under normoxia, after treatment with rmIL-17, %MT was not changed, while RVHI and the RV/body weight ratio were increased, indicating that IL-17 directly induced right ventricular hypertrophy. In a time-course study, the mice were exposed to hypoxia for 0, 1, 2, 3, 4 weeks, respectively. We found that the expression of IL-17 was gradually upregulated in RV tissue in a time-dependent manner after one week of hypoxia exposure, especially at the third and fourth week. Cardiomyocyte hypertrophy and apoptosis were observed after the exposure of the mice to hypoxia for four weeks, rmIL-17 further aggravated the hypoxia-induced cardiomyocyte hypertrophy and apoptosis. The expression of p-STAT3 in the IL-17-deficient mice was lower than in the wild-type mice. In vitro, IL-17 inhibited cardiomyocyte viability and induced cardiomyocyte apoptosis via STAT3 under both normoxic and hypoxic conditions. Conclusions These findings support a role for IL-17 as a mediator in the pathogenesis RVH, which might be considered as a potential novel anti-inflammation therapeutic strategy or diagnostic biomarker for RVH.

Investigating the Role of Farnesoid X Receptor in Heme Biosynthesis and Ductular Reaction

Bile acids (BAs) have gained traction not just as emulsifiers of fat, but also as hormones. Nuclear receptor Farnesoid X receptor (FXR) is the master regulator of BAs and can also control glucose and lipid metabolism. We examined if FXR contributed towards heme biosynthesis and induction of a ductular reaction. Male and female whole body Fxr knockout (FxrKO) mice, as well as liver- and intestine-specific knockouts (LFxrKO and IFxrKO, respectively) were treated with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC, a ferrochelatase inhibitor) for two weeks. At the end of the two weeks, mice were fasted for four hours and euthanized. All groups of mice had lost a similar percentage of body weight when fed the DDC diet. However, female FxrKO mice had significantly increased liver to body weight ratio, while male FxrKO mice had significantly decreased liver to body weight ratio when fed the DDC diet compared with their wild type counterparts. Serum liver injury markers were analyzed and liver histology and changes in genes involved in the heme biosynthesis pathway were examined. Both male and female whole body FxrKO livers had decreased ductular reaction with minimal bile plugs (porphyrin accumulation) compared with their wild type counterparts. LFxrKO mice mimicked diminished ductular reaction, while IFxrKO mice exhibited severe ductular reaction similar to that of wild type mice, indicating that the ductular reaction is dependent on hepatic FXR. ChIP-Seq for FXR revealed binding peaks in the heme biosynthesis genes, Alas1, Alad, Uros, and Fech, suggesting that FXR may act as a transcription factor for these genes. Further investigation revealed that Pbgd gene expression was increased, while Fech gene expression was decreased in female FxrKO mice compared to wild type mice. In male mice, Pbgd, Uros, Urod, and Cpox gene expression was increased in the absence of Fxr. In conclusion, Fxr is necessary to mount a ductular reaction and plays a key role in heme biosynthesis in the liver.

MO337UNILATERAL NEPHRECTOMY OVERCOMES PROGRESSION TO CHRONIC KIDNEY DISEASE AFTER ACUTE INJURY IN MICE BY STIMULATING PROLIFERATION OF RENAL PROGENITOR CELLS

Background and Aims Acute kidney injury (AKI) is a global health concern with an incidence of 13.3 million patients per year, and increasing. AKI is recognized as an important risk factor for the development of chronic kidney disease (CKD). A crucial aspect for successful renal recovery after AKI is an efficient proliferative response of surviving tubular epithelial cells (TECs). Recently, we established a murine model in which the functional and histological recovery of a single kidney, injured by ischemia, is enhanced by removal of the unharmed contralateral kidney; a phenomenon termed nephrectomy-induced recovery. The renal epithelial reparative response in this unique physiological model has not been investigated, yet can provide new insights in unlocking the inherent regenerative potential of the renal epithelium. Method AKI was induced in R26RtdTomato and PAX2/Confetti mice by left unilateral ischemia/reperfusion (UIRI) for 21 min at 34°C, after which either right nephrectomy (Nx) or no Nx was performed 3 days later. Mice were euthanized 6 weeks and 28 days after UIRI, respectively. At week 6, kidneys were weighted and renal function was assessed by serum creatinine. At 28 days, renal tissue of Pax2/Confetti mice was collected to perform renal progenitor cell lineage tracing experiments by immunofluorescence and confocal microscopy. Results When nephrectomy was performed after UIRI, left kidney-to-body weight ratio did not change significantly over time, whereas, when no nephrectomy was performed, left kidney-to-body weight ratio gradually declined from 7,84 ± 0,48 mg/dl at day 3 till 3,26 ± 0,51 mg/dl at week 6, indicating severe atrophy in the injured left kidney. This loss of renal mass was associated with a significant increase in serum creatinine (1,76 ± 0,13 mg/dl) as compared to control (0,21 ± 0,12 mg/dl), whereas with nephrectomy, renal function fully restored. Clonal analysis in PAX2/Confetti mice revealed that nephrectomy after UIRI led to a significant increase in proliferating (i.e. clonogenic) Pax2+ progenitor cells, resulting in more multicellular clones as compared to un-nephrectomized controls. Conclusion Nephrectomy after UIRI overcomes chronic loss of renal mass and function within the investigated 6-week time frame. This study is the first to demonstrate that nephrectomy stimulates clonal expansion of renal progenitor cells in an injured kidney, beyond that observed for spontaneous repair after UIRI. Insight in the signaling mechanisms may reveal new therapeutic approaches to incite the inherent renal regeneration potential.

MO019MATERNAL UNDERNUTRITION AMELIORATES CYST GROWTH IN ADPKD MODEL MICE*

Background and Aims Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the progressive development of kidney and liver cysts. The major factors predicting disease progression in ADPKD are total kidney volume, genotype, age, sex. Recently, several studies have suggested that dietary intervention might be a potential treatment to prevent ADPKD progression. On the other hand, it has been reported that low-birth weight infants because of maternal global nutrient restriction are at increased risk for hypertension, type 2 diabetes, and metabolic syndrome and chronic kidney disease. However, little has been reported on relationship between maternal undernutrition and cyst formation in ADPKD. Therefore the purpose of this study is to clarify whether maternal undernutrition is associated with progression in ADPKD. Method We used Pkd1 conditional knockout mice (Pkd1flox/flox･Mx1-Cre mice). The offspring of dams given food ad libitum (control(CON)) and those subjected to nutrient restriction throughout pregnancy (food restriction (FR)) were examined. In FR, nutrient-restricted mothers were given about half amount of food by control mice during pregnancy. After delivery, food for children was given ad libitum. Mice were injected with polyinosinic-polycytidylic acid for 6 consecutive days from postnatal day 5 (P5) to P10 to inactivate Pkd1. We analyzed the phenotype of cystic kidneys by kidney/body weight ratio (2KW/BW), and cystic index (CI) which was defined as the percentage of areas occupied by cysts at P20, 35, 56. We carried out a series of analyses by kidney/body weight ratio, liver /body weight ratio or cystic index (CI) which was defined as the percentage of areas occupied by cysts. Elastica-Masson staining was performed for analyzing tissue fibrosis. The fibrotic index (FI) was expressed as the (fibrotic area/ total non-cystic area) ×100 (%). For evaluation of glomerular hypertrophy, glomerular area was measured in PAS-stained kidney sections using Image J software. The assay of renal function was performed by using UN-ML kit. We also performed western blotting of signaling pathway of proliferation by using whole kidneys. Data are shown as mean ± SEM. Two-tailed Student’s t-test was performed for comparing two groups. Results Food restriction of pregnant dams reduced birth weight. (FR 1.26±0.16 g vs CON 1.55±0.11g). However, FR showed rapid gain weight. There was no significant difference after P20. There were no difference between two groups in 2KW/BW, serum blood urea nitrogen (BUN) levels, CI of kidney and liver until P35. At P56, 2KW/BW was significantly greater in FR (4.59±0.52%) than in CON mice (2.91±0.98%; p＜0.01). CI of both kidney and liver was significantly higher in FR than in CON (Kidney : FR58.1±2.04% vs CON46.3±3.04%; p＜0.05) (Liver : FR11.1±1.41% vs CON 4.89±0.61%; p＜0.001). BUN levels elevated in FR (FR 58.1±5.76mg/dL vs CON 46.3±5.11mg/dL; p＜0.05) .FR showed glomerular sclerosis with PAS staining. Mean Glomerular volume was significantly increased in FR compared with CON (P35: FR 5625±419μm2 vs CON 3255±433μm2; p＜0.001, P56: FR 5780±1195μm2 vs CON 3756±266μm2; p＜0.001) . FR group showed significantly greater fibrosis in kidney (P35: FI 15.3±2.04% vs CON 11.1±1.41%; p＜0.01,P56: FI 42.1±3.2% vs CON 30.8±3.89%; p＜0.001). In western blotting analysis, MAPK pathway and mTOR pathway were suppressed in FR compared with CON at P20. In contrast, MAPK pathway and mTOR pathway were upregulated in FR compared with CON at P56. Conclusion Maternal undernutrition accelerates disease progression via kidney fibrosis, MAPK and mTOR pathway.