Phyllanthusemblica fruits in the reversal of remodeling changes in pressure overload left ventricular cardiac hypertrophy (LVH) and age-associated cardiac dysfunction in male Wistar rats. Six groups (aging groups) of 3 months old animals were given either AR or ghee and honey (GH) orally; seventh group was untreated. Ascending aorta was constricted using titanium clips in 3 months old rats  and after 6 months, AR or GH was given for further 12 months to two groups; one group was untreated. Histology, gene and protein expression analysis were done in heart tissues. Chemical composition of AR was analyzed by HPLC, HPTLC and LC-MS. AR intake improved (P < 0.05) cardiac function in aging rats and decreased LVH (P < 0.05) in AC rats as well as increased (P < 0.05) fatigue time in treadmill exercise in both groups. In heart tissues of AR administered rats of both the groups, SERCA2, CaM, Myh11, antioxidant, autophagy, oxidative phosphorylation and TCA cycle proteins were up regulated. ADRB1/2 and pCREB expression were increased; pAMPK, NF-kB were decreased. AR has thus a beneficial effect on myocardial energetics, muscle contractile function and exercise tolerance capacity.We evaluated the cardioprotective effect of Amalaki Rasayana (AR), a rejuvenating Ayurvedic drug prepared from  Rejuvenation and remodeling strategies comprise the ‘Rasayana chikitsa’ (rejuvenation therapy) in Ayurveda. Rasayana  group is a class of medicinal plants in Ayurvedic pharmacology used for this purpose. ‘Rasayana’ drugs refurbish the neuronal, endocrinal and immune systems and are considered to prevent ageing, re-establish youth, strengthen life, brain power and prevent diseases, thus enhancing bodily resistance to all kinds of injury3.Ayurveda which signifies the science of long life, is one of the ancient (>5000 years old) systems of medicine in India. Health promotion, disease prevention and rejuvenation approaches are used in this system of medicine through dietary and therapeutic means and both approaches can slow aging and invigorate functions of the body’s organsPhyllanthusemblicaor Embilicaofficinalis; Family: Euphorbiaceae, Aurvedic name: ‘Amala’) is an Indian traditional Ayurvedic drug used as a rejuvenating medicine in aging conditions. The fruits of Amala commonly used in Aryuveda are assumed to enhance defense against diseases. Ayurveda literature mentions Amalaki to have a beneficial role in cancer, diabetes, liver and heart diseases, gastric ulcers and various other disorders. Amalaki rasayana has antioxidant, immunomodulatory, antipyretic, analgesic, cytoprotective, antitussive and gastroprotective actions. It is also used for memory enhancement and for lowering blood cholesterol levels. Reports from various scientific studies suggest that its consumption also protects tissues against radiation damage7. In recent studies, AR was seen to increase the median life span and starvation resistance in Drosophila melanogaster8 and increase the levels of apoptosis inhibitory proteins (DIAPs) and decrease caspases and levels of Rpr, Hid or Grim (RHG) pro-apoptotic proteins in the eye disc and salivary glands of AR treated Drosophila9. AR fed aged rats were also found to have increased genome stability in astrocytes and neurons of the cerebral cortex10.Amalaki rasayana/AR  and inhibit and attenuate calcineurin activation11. Bhattacharya SK et al. studied the effect of tannoid principles of fresh juice of emblica fruits on ischemia-reperfusion (IRI)-induced oxidative stress in the rat heart. Administration of tannoid principle prevents IRI-induced effect, when given orally twice daily for 14 days12. Rajak S et al. discovered that chronic administration of Amala improved antioxidant defense of myocardium in IRI induced through oxidative stress. Their results indicate that long term administration of fresh Amala fruit homogenate (500 and 750 mg/kg) can augment endogenous antioxidants and protect rat hearts from associated oxidative stress in IRI13. Bhatia J et al. evaluated the effect of hydroalcoholic lyophilized extract of emblica in rats with hypertension induced by deoxycorticosterone acetate or 1% sodium chloride high salt (DOCA/HS) administration14. They reported that Emblicaofficinalis reduces oxidative stress and prevents development and progression of hypertension by modulating levels of serum NO, activated eNOS endogenous antioxidants, and electrolytes. Studies by Yokozawa et al. indicate that Amala may attenuates oxidative stress and may prevent hyperlipidemia associated with aging15.In recent times, there is mounting interest in the use of natural products as secondary medicines for cardiovascular disease. Juan Guo in vivo studies have been previously done to evaluate the cardio protective effect of AR in pressure overload hypertrophy and cardiac function in aging animals.These studies prompted us to evaluate the effect of AR in the reversal of remodeling changes in pressure overload left ventricular cardiac hypertrophy and age-associated cardiac dysfunction in rats. No We observed that long term oral intake of AR improves cardiac function in aged rats as well as in rats with pressure overload left ventricular hypertrophy. The functional improvement was associated with enhanced myocardial contractile function and mitochondrial bioenergetics.Results of qualitative solubility analysis of AR and a mixture of ghee and honey (GH) used as carrier in AR in different solvents are given in Supplementary Table HPTLC profiles of samples of the finished formulation revealed the presence of gallic acid and ellagic acid , norepinephrine sulfate and vitamin metabolites, as identified from online software XCMS for metabolomics study  for up to 72 hours , QT interval were not observed in any of the animals. A mean blood pressure of 116–120 mmHg was noted in rats of all the groups. Heart rates were also within normal limits in all the rats.P < 0.05) improved in AR administered group of rats, when compared to GH treated/control rats. The animals which received larger doses of AR had more beneficial effects than those which received 250 mg/kg body weight  and left ventricular ejection fraction (LVEF) were found to be significantly  increased in AR administered rats, when compared with rats of other experimental groups. Fatigue times were more in the animals which received larger doses of AR than those which received 250 mg/kg body weight  ratios in AR, GH administered and control rats are given in Supplementary Table To observe toxicity or other adverse changes after AR administration, we also recorded absolute and relative weights of lungs, livers and kidneys and did histopathology of tissues from these organs of rats after sacrificing at the age of 21 months. Absolute tissue weight, tissue weight/body weight ratio and histology did not reveal significant weight gain/loss, denuding bronchiolitis or perivascular cuffing in the lungs, steatosis or hepatiits in liver or glomerular sclerosis or tubular lesions in the kidney  was 125–130 mmHg in rats of all the groups. Mean heart rates (HR) were also within normal limits in all the animals.P < 0.001) and significant decrease in LVIDd, LVIDs (P < 0.001) at the end of 6 months (Supplementary Table P < 0.05) decreased the IVSd and LVPWd at the end of 21 months (12 months after treatment with AR) in rats with left ventricular hypertrophy when compared with GH administered rats with left ventricular hypertrophy  increased in the rats with left ventricular hypertrophy and were administered AR  different among AR, GH administered or control rats  were significantly  analysis from trichrome stained heart sections also revealed mild interstitial fibrosis in AR treated rats while all the rats of GH treated or control rats had moderate degree of myocardial fibrosis Fig. .We did not find significant differences in BNP levels in AR administered group of rats when compared with other groups. Mean BNP levels were however, >200 pg/ml at the end of 18 months after constriction in rats which underwent constriction of aorta Fig. .P < 0.05) increase in the expression of TCA driving enzymes ; OXPHOS complex enzymes , FAO , muscle contraction  and antioxidant defense  proteins  analysis from heart tissue samples of control (untreated), AR and GH administered rats. Protein expression profile revealed that hearts of AR treated rats have increased expression of proteins regulating the tricarboxylic acid (TCA) cycle, oxidative phosphorylation (OXPHOS), fatty acid oxidation (FAO), cardiac muscle contraction, antioxidant defense and mitochondrial proteins Fig. . LC-MS pins Fig. .Figure 3P < 0.05) up regulated in AR administered rats while PLN and CAT1 were down regulated or unchanged respectively  analysis. Consistent with the proteomic results, SDHA, COXIV, ATP5a1, SERCA2, TroponinT, Myh11 Fig.  and SOD1ely Figs  and 5a,cely Figs .Figure 4P < 0.05) increase in beclin-1 and decrease in p62 were also seen, suggesting autophagy induction in AR administered rats. We also observed a significant (P < 0.05) decrease in the levels of AMPKα phosphorylation in heart tissues of rats administered AR suggesting an energy efficient myocardium  decrease in the expression of NFkB, a transcription factor which regulates stress response signaling in hearts of rats administered AR  increased levels of CREB-1 (Ser133) phosphorylation , sulfate derivative of norepinephrine and putative arachidonic acid derived anti-inflammatory metabolites, in AR. Previous studies have reported that these metabolites play central roles in the regulation of myocardial bioenergetics, contractile, myocardial inefficiency and dysfunctional excitation-contraction coupling and hemodynamic function23. Presence of these components in AR could possibly contribute to improvement in cardiac function in rats administered with AR. We have observed gallic acid and ellagic acid as the major components of AR; vitamin C was present in only relatively small amounts in AR. In a recent study, Vishwanatha et al., also reported the presence of gallic acid as the major component (>42% abundance) of Amalaki rasayana. Vitamin C was only 1.8% in AR. They found that AR administration stably maintained the double strand break repair (DSBR) without altering the nucleotide excision repair or base excision repair in aged human individuals3. Gallic acid, ellagic acid and vitamin E are known to increase the antioxidants status in the body25. Abundance of these components in AR could lead to increase in the antioxidant enzymes SOD1 or 2 as well as Gpx and decrease in NF-kB expression. Both Vitamin A and D3  have been shown to attenuate cardiac remodeling associated changes with hypertrophy26. Presence of these components in AR could have contributed to decrease in degrees of hypertrophy and fibrosis or increase in anti-hypertrophy markers  in AR administered animals. Nor-adrenaline sulfate present in AR may contribute to increase in ADRD1/2, pCREB-1 or exercise tolerance as reported previously28. Arachidonic acid metabolite (5(S), 6(S)-epoxy-15(S)-hydroxy-7E, 9E, 11Z, 13E-eicosatetraenoic acid), play important roles in optimal metabolism and in reducing heart disease risk30. This component in AR possibly aids in maintaining regulation of OXPHOS pathway, pAMPK, antioxidant enzymes status and decrease in NF-kB expression. The effects observed after AR treatment may be due to synergistic effects of the components. The doses of individual components in AR are small and we have not explored whether these small doses of each component separately would produce the beneficial effects.AR administration increases the expression of antioxidant defense and β32. We found significant improvement in LVEF and LVFS in the aging animals and decrease in IVSd and LVPWd in animals with left ventricular hypertrophy secondary to aortic constriction which were administered AR. Age related decline in exercise tolerance and physical deconditioning are well established34. AR significantly improved the fatigue time in treadmill exercise in aging rats and rats with severe left ventricular hypertrophy.Previous studies have shown cardiac structural changes associated with aging. These changes consist of increase in cardiomyocyte size, increased septal thickness and increased internal dimension of the left ventricle. They contribute to contractile dysfunction in the aging heart37. AR is found to enrich OXPHOS, FAO, mitochondrion regulatory proteins and up regulate expression of OXPHOS complexes such as SDHA, COXIV, and ATP5a1 in cardiac tissues.We have explored the molecular mechanisms of how AR possibly contributes to improvement in cardiac function. Previous studies suggest that altered mitochondrial dynamics or reorganization of respiratory chain complexes, oxidation of mitochondrial proteins and reduced biogenesis contribute to decline in efficiency of mitochondrial bioenergetics associated with aging39. In cardiac tissues of AR administered rats, phosphorylation levels of AMPKα, an indicator of sufficient OXPHOS function was decreased, which suggests that these rats have energy efficient hearts.AMPK is a nutrient sensor transcription factor which regulates cellular energy metabolism and aging through multiple mechanisms. AMPK activation shuts off mTORC, indicates a high -AMP/ATP-low stage in aged myocardium and contributes to energy inefficiency40. In our study, p53, SOD1, SOD2, GPX1 levels were significantly increased in the hearts of AR administered rats. These changes indicate improved antioxidant defense upon AR administration. Nuclear factor –kappaB (NFkB), a redox sensitive transcription factor is known to be up regulated during stress response signaling and in turn reduces the expression of SOD1 and SOD2 genes in the mitochondria42. Expression of NFkB was decreased in the hearts of AR administered rats. This observation further strengthens the possibility that AR improves the antioxidant defense in aged heart.The free radical theory of aging proposed that gradual decline in mitochondrial function with aging contributes to impaired electron transport chain (ETC) function and reduced ATP generation. Deficient ETC results in increased production of ROS, which in turn intensifies the mitochondrial dysfunction and overall cell damage44. It is widely known that beclin-1 is required for initiation of the autophagosome formation. LC3II accumulation indicates autophagy induction while p62 is degraded by autophagy process45. There was increased expression of autophagy markers LC3 and beclin-1 as well as decrease in the expression of p62 (marker for autophagy induction) in the hearts of AR administered rats, which suggests the effect of AR on a healthy cellular or tissue aging process.Several studies have shown that either accumulation of fragmented mitochondria or decline in clearance of damaged organelle (autophagy) or both, contribute to increased mitochondrial degeneration which drives the pathological changes during aging2+-transport or SERCA activity, altered expression of calmodulin (Ca2+ binding protein), and sarcopenia are the reported critical changes during cardiac remodeling during aging47. Increased expression of SERCA2, Calm3, TroponinT, Myh11, and decreased expression of PLN genes were seen in the hearts of AR administered rats. The increased muscle contractile performance could be the contributory factor in increased fatigue time in the AR administered rats.AR also seems to improve the muscle contractility in the aged heart. Impaired cardiac muscle excitation – contraction coupling, decreased ATP dependent sarcoplasmic reticulum Ca48.We analyzed the expression of selected membrane receptors (β1/2 adrenergic receptor (ADRB1/2), voltage dependent Ca2+ channel (CACNA1S), Na+-Ca2+ exchanger/NCX) whose activation can regulate Ca2+ regulation, muscle contraction or metabolic activity in the hearts of rats in our experiments. Increased expression of β1/2-adrenergic receptor especially ADRB1 gene, was seen in the hearts of AR administered rats which indicates activation of these receptors. An increased phosphorylation of CREB-1, a downstream target of ADRB activation was also seen.Human and animal studies have revealed a decline in beta –adrenergic receptor sensitivity, or receptor density which contribute to decrease in cardiac contractility, SR dependent Ca2+ signaling and ejection fraction in the aged heart51.Our study thus throws light on the possible mechanisms by which AR intake results in improved exercise tolerance. These mechanisms include improved mitochondrial bioenergetics through transcriptional regulation of mitochondrial OXPHOS and enhanced muscle contractility through activation of endogenous β-adrenergic receptor 1 and 2 (β-AR). The effects of AR seem to have similarities to those of standard Western drugs such as digitalis and nor-adrenaline and adjunctive agents such as L-carnitine and Coenzyme Q52. Mertens-Talcott et al., (2006) reported urolithin A, hydroxyurolithin A, urolithin A-glucuronide, urolithin A and dimethyl ellagic acid-glucuronide as metabolites of EA in human urine samples53. Cardiac effects of these metabolites are unknown.Our study has some limitations. AR was administered to animals for only 5 days a week. A continuous administration for 7 days could produce a different response. For preparation of AR, we have followed the procedure specified in the Ayurvedic Text Charaka Samhita. The rationale for repetition of some of the steps in the procedure such as trituration, are unknown and needs further validation. Also, we did not perform pharmacokinetic analysis for AR. Previous pharmacokinetic analysis of gallic acid, the major component of AR has revealed the 4-O-methylgallic acid (4OMGA) as the major metabolite in human plasmaIn summary, our data suggest that AR could be an important adjuvant for improvement of cardiac muscle contractility and mitochondrial energy efficiency in aging and hypertrophied heart.All animal experiments were carried out with the approval of the Institutional animal ethics committee (IAEC) in Rajiv Gandhi Center for Biotechnology (RGCB) under the protocol no. IAEC/150/CCK/2012. Animal experiments were conducted by strictly following the rules and regulations of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India.Amalaki rasayana and GH were prepared and supplied by Arya Vaidya Sala, Kottakkal, Kerala, India. The fresh green Amalaki was procured from traders who source it from the Sathyamangalam area of Tamil Nadu . The dried variety was procured from traders in Madhya Pradesh who, in turn, source it from Chattisgarh  and from Madhya Pradesh . Fresh gooseberries used for preparation of juice were harvested in November, 2012 and March, 2013. Gooseberries used to prepare the powder were harvested in November, 2011. The procedure used for preparation is given below and has been described earlier9.Step 1. Dried gooseberry fruits (Phyllanthusemblica) were pulverized by a Tyco pulverizer to obtain powder (80 mesh).Step 2. Fresh gooseberries were crushed by using a juice extractor to obtain the juice.Step 3. The powder (product of step1) was blended in the gooseberry juice (product of step 2) in 1:1 ratio and dried for 24 hours at 55 °C under 700 mmHg by using a vacuum tray drier. The dry mass was then pulverized.bhavana (trituration). This is a procedure prescribed for the purpose of potentiating the basic activity of the ingredient herb and it involves mixing the powder with a juice medium, then drying it and again going through the cycle repeatedly. This is the standard procedure recommended in the preparation of Amalaki rasayana54.Steps 2 and 3 were repeated 20 times. The 21 times repeated step is called in Ayurvedic texts as Step 4. The powdered mass of amalaki (obtained after completion of 21 trituration cycles) was blended with honey  and Ghee  in a 1: 2:0.5 ratio. Finally, a thick pasty mass of Amalaki rasayana was obtained as described previously by Dwivedi et al., 20128. Composition of ghee used is provided in the Supplementary Table 55.The ancient Ayurvedic text ‘Charaka Samhita’ mentions method of preparation of AR and we have followed the method prescribed in this text. Honey and ghee are added to the triturated Amalaki powder as ‘anupana’ (carrier or adjuvant), which is considered as a medium to improve acceptability and palatability and also to help absorption of the main ingredientGH was constituted with a mixture of ghee and honey as (mixed in a fixed proportion (2: 0.5)) used in the preparation of Amalaki rasayana.et al., 2012. Briefly, Centre for Medicinal Plants Research, Kottakkal and the Quality Assurance (QA) department of Arya Vaidya Sala,  authenticated the Amala fruit and powder.Quality assurance and control assays were performed by standard protocols as described by Dwivedi http://www.ayurveda.hu/api/API-Vol-1.pdf)56. Ayurvedic Pharmacopoeia of India contains the Official Standards to be adapted and followed for Ayurvedic preparations. HPTLC profiles of raw Amalaki fruits, Amalaki powder, samples at the conclusion of 1st, 10th, 15th and 21st steps of trituration and the finished formulation revealed the presence of gallic acid and ellagic acid compared with the respective standards.Ayurvedic Pharmacopoeia of India (API) certified that macroscopic (shape and taste of fruits) and microscopic  characteristics of Amala were equivalent with the specified standards  acetonitrile: water (20:80 v/v) and (70:30 v/v), (ii) methanol: water (20:80 v/v) and (70:30 v/v), (iii) ethanol: water (20:80 v/v) and (70:30 v/v) and diethyl ether:water (20:80 v/v) and (70:30 v/v). The analysis was performed by adding 10 mg of each preparation in different solvents to observe the maximum solubility. 20% ethanol in water (20:80 v/v), was observed as a good solvent for both AR and GH. Further, after assessment of solubility, RP-HPLC profile was analyzed for both AR and GH.AR and GH were analyzed using Waters HPLC equipped with symmetry C18 column , 2707 auto sampler and 2489 UV/Visible detector. Breeze software  was used for monitoring and processing output signal. Elution was done at a flow rate of 1 ml/min using mobile phase comprising of Solvent A [Water: acetic acid (ratio of 99.9:0.1 v/v)] and solvent B as acetonitrile. The samples were eluted by gradient mentioned in Table The relative hydrophobicity of elution system shifts from more hydrophobic compounds to less hydrophobic compounds. Thus, for further identification of components of AR, LC-MS analysis was done.254) plate and separated using the mobile phase Toluene: ethyl acetate: formic acid: Methanol (6: 6: 1.6: 0.4) ratio. Step 3: after TLC fractionation of AR, three different components  were separated on silica plate. Step 4: separated components were scraped from plate under UV light, and all the three components were dissolved in methanol (100%) and were lyophilized. Step 5: the lyophilized fractions (dissolved in acetonitrile) were given for LC-MS analysis using the instrument . MassLynx and PLGS v2.5.3 were used as software for data acquisition and for raw data processing respectively. Step 6: after getting the data from the LC-MS, we uploaded the data in XCMS online software for metabolite identification.Step 1: One gram of AR was dissolved in 20 ml (20% ethanol in water) in a conical flask at 4 °C under overnight stirring. Step 2: From the solution, 10 μl volume was loaded on TLC  were obtained from American Type Culture Collection. The cells were maintained in DMEM (95%), Fetal bovine serum (5%), Penicillin/Streptomycin (U/ml) at 37 °C in 5% CO3 cells per well at 37 °C in CO2 incubator. After overnight incubation, cells were incubated with or without AR in different doses  for 24 h, 48 h, or 72 h. After completion of incubation time, the medium was removed and 20 μL of MTT (5 mg/mL in PBS) was added with fresh medium. To dissolve the formazan crystal formed, 100 μL of isopropanol was added with mild agitation at room temperature for 1–2 minutes. Spectrophotometric absorbance was measured at 570 nm in an ELISA microplate reader (BioRad). The percentage of cell viability was calculated by the following formula: Viable cells % = (OD of AR-treated cells/OD of untreated (MTT + Isopropanol only) cells) × 100.-2,5-diphenyltetrazolium bromide) assay. To assess the safety/toxicity profile of AR, MTT assay was performed in H9c2, the rat cardiomyoblast cells. Cells were sub cultured in 96-well microtiter plates at a density of 5 × 10ad libitum access to water.Three-month- old male Wistar rats, (180–200 g) were used. The animals were housed in 12-hours day/light cycle with temperature and humidity controlled room. Rats were fed with rodent synthetic chow diet and had 2. To induce ‘pressure overload’ cardiac left ventricular hypertrophy, the ascending aorta was constricted (AC) using a titanium clip 51. Pressure gradient was recorded using trans- thoracic two dimensional color Doppler analysis to ensure physiologic constriction of the aorta. Sham operated rats underwent similar surgical procedure as in rats with constriction of aorta except for constriction of aorta. Left ventricular hypertrophy was consistently observed during echo studies in rats after 8 weeks of aortic constriction.Rats were anesthetized with 3% isoflurane with 100% OTwo sets of experiments , 56 male rats (3 months of age) were divided into seven groups; three groups (each group having 8 rats each) were given AR . Another 3 groups (each group having 8 rats each) were given GH  while the seventh group was left untreated as controls (n = 8). All these rats in the 7 groups were allowed to grow up to 21 months of age. Since effects of AR on cardiac function has not been investigated previously, we tried three different doses . We found 500 mg/kg body weight dosage optimally effective.13. In experiment II (ascending aorta constricted–AC groups), ascending aorta was constricted using titanium clips to induce left ventricular hypertrophy in 24 rats (3 months old). After the rats developed left ventricular hypertrophy, AR  or GH  was given to 2 groups of rats. Another 8 rats (AC) did not receive either AR or GH.This dose was considered for experiment 2. 500 mg/kg body weight dosage was selected as an optimum dose also because of the traditional use in humans in Ayurveda practice and also based on previous animal studiesOur analysis of AR revealed that a 500 mg/kg body weight dosage of AR would have 7.5 mg gallic acid (GA) and 2 mg ellagic acid (EA) as daily dose. All the animals were observed daily for food and water intake as well as for any untoward symptoms. Body weights were regularly recorded.AR or GH was administered orally to the aging group of rats each day morning for 5 days a week until 21 months of age. The rats in AC group were administered AR or GH orally each day for 5 days a week, for 12 months after the rats developed left ventricular hypertrophy (9 months of age) as assessed by echocardiography. The experiment was continued until the AC group rats attained 21 months’ age.32 we performed echocardiography studies. Echocardiography was performed in all the rats at 3 months of age before starting the experiments (prior to administration of AR), 12 months of age (9 months after start of AR administration) and 21 months of age (18 months after start of AR administration) in the aging group. In AC group, echocardiography was performed in all the rats before the start of the experiment (3 months of age) and after constriction of aorta at intervals of 3, 6 (6 and 9 months of age respectively) and 21 months (12 months after initiation of administration of AR).To investigate the effect of AR on age associated changes in left ventricular dimensions and ejection fraction associated with agingOne month prior to sacrifice of animals, all the rats were trained on a motorized treadmill (Columbus) with individual Plexiglas lanes for 30 min, 5 days/week. During the first week of training, animals were acclimatized to the treadmill by gradually increasing the running speed , without inclination while frequency and number of shocks were fixed each time at gradually increasing speeds. At the end of two weeks of training, exercise tolerance was assessed by following the standardized protocol . Fatigue time versus distance travelled was measured in triplicate for all rats  in both the aging and AC groups.Assessment of heart failure was carried out at the end of the experiment, by estimation of serum levels of brain natriuretic peptide (BNP). BNP assay was done following the manufacturer’s protocol with BNP -32 rat ELISA kit (Abcam).Animals were sacrificed by euthanasia and the hearts were collected.10% neutral buffered formalin was used for fixing the 5 mm thick cross sections above the apex and processed for paraffin embedding following standard protocol. Tissues were also collected from the heart for proteomic profiling by Liquid chromatography and mass spectrophotometry, qRT-PCR and Western blot. These tissues were collected in RNAlater, kept at room temperature for 12 hours and then stored in −80 °C until analysis.min, μm) and major cell diameter , comparable to the cardiomyocyte length, were measured as parameters to quantify the cardiomyocyte size from trichrome stained sections. Longitudinally cut cardiomyocytes were selected for Dmaj and multiple measurements were made from each of the anterior, septal, lateral, and inferior wall in LV sections. Cross sections of cardiomyocytes were taken to measure Dmin. Well defined cell membrane and visible nuclei were the criteria for selection of myocytes . Hematoxylin and Eosin and Masson trichrome , stained sections were examined under a Nikon Eclipse 55i microscope for assessment of myocyte and vascular changes and fibrosis respectively. To quantify the cardiomyocyte diameter and fibrosis, we have used software NIS element (Nikon). Hypertrophy was measured by calculating the HW/BW (mg/g) ratios and cardiomyocyte dimensions. Minor cell diameter  and fibrosis (area %) were calculated based on the analysis of perivascular and interstitial fibrosis in 15 microscopic fields from each section of left ventricle (n = 6) by using software NIS element from Nikon 55i microscope. Semi-quantitative scores of 0, 1, 2 and 3/4 were assigned for normal, mild, moderate and severe fibrosis. Percentage fibrosis was calculated as percent relative blue staining to total myocardial area from the quantified RGB images.Sections from paraffin embedded tissue blocks were deparaffinized and rehydrated. After retrieval of antigenicity in tissue sections, Super Sensitive Polymer-HRP IHC Detection System/DAB  kit was used for further steps. Mouse monoclonal antibody to smooth muscle myosin heavy chain 11 (ab683) was used in 1:100 dilutions with 3% BSA in PBS. After washing with PBS, sections were incubated for 1 hour with secondary antibody conjugated to horse radish peroxidase (HRP). Then the sections were stained with hematoxylin (Sigma-Aldrich) for nuclei and mounted in DPX . Intensities of immunostained images were quantified by using Nikon NIS elements software (Japan). The relative intensity of staining in the myocardium was corrected for background, normalized with GAPDH and expressed as fold change relative to untreated samples (N = 6).TM SF surfactant in 50 mM NH4HCO3 buffer . Total protein content was estimated by Bradford assay. Peptide was generated for each sample (100 µg of protein) using in-solution trypsin digestion. Solutions of digested peptide were centrifuged at 14000 rpm for 12 minutes and supernatant was stored at −20 °C until LC/MS/MS analysis.Heart tissues were ground, homogenized with a pestle and mortar in liquid nitrogen and the lysate was prepared in 0.3% RapiGestPeptide samples were analyzed by using a nano ACQUITY UPLC® System  coupled to a Quadrupole-Time of Flight (Q/TOF) mass spectrometer  controlled by MassLynx4.1 SCN781 software . Peptides eluted from the nano LC were monitored by SYNAPT® G2 High Definition MS™ System . Three technical replicate runs were performed for each sample.Rattus novergicus database from NCBI. Protein identification was performed by setting the parameters for each peptide such that at least one fragment ion match and a protein was required to have at least three fragment ion matches, or at least two peptide matches for identification. Precursor and fragment ions were defined by setting the Mass tolerance at 10 and 20 ppm respectively. Oxidation of methionine and carbamido-methylation of cysteine were chosen as variable modification and fixed modification respectively. Auto-normalization of PLGS was done to normalize the dataset and label-free quantitative analyses were carried out and compared with normalized peak area/intensity of identified peptides between the samples. Number of peptides, score, and sequence coverage parameters were identified for each protein. The reference sequence identifications (RefSeq.) obtained after the PLGS analysis was converted into gene symbols by Biological Database Network (BioDBnet). Gene symbols were categorized into different biological functions by using a bioinformatics tool, Database for Annotation, Visualization and Integrated Discovery (DAVID). Statistical analysis and graphical representations were performed through MS-Excel 2010 and GraphPad Prism (6.0).LC/MSE data were analyzed by using ProteinLynx Global SERVER™ v2.5.3  which employs protein identification as well as relative quantification. Database search was performed using qRT-PCR (Quantitative reverse transcription polymerase chain reaction): Heart tissues were homogenized in Trizol (Life technologies 15596-018) with a pestle and mortar. After confirmation of the quality, RNA was reverse transcribed into complementary DNA followed by gene amplification through qRT-PCR using the software SDS in Bio-Rad RT-PCR machine. After confirmation of primer efficiency in all the experiments, primer specificity (single product amplification) was analyzed by melting-curve analysis. Target mRNA expression was quantified, normalized to the GAPDH and fold change calculated by 2−ΔΔCt method. mRNA expression results were represented as fold difference in target gene amplification in tissues of AR/GH administered rats, relative to amplification in tissues of untreated rats (N = 6). Details of primers used are provided in Supplementary Table Immunoblot analysis: Heart tissues excised from the rats during sacrifice were snap - frozen in liquid N2 and stored at −80 °C. Heart tissues were ground homogenized with a mortar and pestle in liquid nitrogen and then lysed with Radioimmunoprecipitation assay buffer (RIPA buffer) using a dounce homogenizer. 40 μg of total protein was loaded for Immunoblot analysis from each tissue lysate. The prepared tissue lysates were processed for Immunoblot analysis. The relative intensity of band in blot was corrected for background, normalized with β-tubulin and expressed as fold difference  in AR, GH administered or control samples relative to the housekeeping protein (β-tubulin). Details of antibodies used are provided in Supplementary Table p < 0.05 as statistically significant.Data were analyzed by software GraphPad Prism (6.0). Results are presented as mean ± SEM unless otherwise indicated. Multi-comparison of data was performed through either a repeated measure one-way ANOVA, one-way ANOVA followed by multiple comparisons with Tukey’s test, repeated measure two-way ANOVA, two-way ANOVA followed by multiple comparisons with Tukey’s test, as indicated in legends for respective figures or tables. We have considered Supplementary Information


AR mutations are more frequently detected in this type of cancer. By analyzing tumor biopsy mRNA from CRPC patients who had developed resistance to CYP17 inhibitor treatment, we have identified a novel nonsense mutation (Q784*) at the ligand binding domain (LBD) of AR, which produces a C-terminal truncated AR protein that lacks intact LBD. This AR-Q784* mutant is transcriptionally inactive, but it is constitutively expressed in the nucleus and can bind to DNA in the absence of androgen. Significantly, our results show that AR-Q784* can heterodimerize with, and enhance the transcriptional activity of, full-length AR. Moreover, expressing AR-Q784* in an AR positive PCa cell line enhances the chromatin binding of endogenous AR and the recruitment of p300 coactivator under the low androgen condition, leading to increased cell growth. This activity of AR-Q784* mimics the function of some AR splice variants, indicating that CYP17 inhibitor treatment in CRPC may select for LBD-truncated forms of AR to restore AR signaling.The standard treatment for prostate cancer (PCa) is androgen deprivation therapy (ADT) that blocks transcriptional activity of androgen receptor (AR). However, ADT invariably leads to the development of castration-resistant PCa (CRPC) with restored activity of AR. CRPC can be further treated with CYP17 inhibitors to block androgen synthesis pathways, but most patients still relapse after a year of such treatment. The mechanisms that drive this progression are not fully understood, but AR activity, at least in a subset of cancers, appears to be restored again. Importantly,  PSA). The standard treatment for PCa is to block transcriptional activity of androgen receptor using androgen deprivation therapy (ADT), which invariably results in the development of a more aggressive form of cancer  with restored activity of AR [Prostate cancer (PCa) is the second leading cause of cancer mortality in American men and is dependent on the activity of androgen receptor (AR) for its initiation and progression. AR is a nuclear receptor transcription factor and is activated by androgen ligands . The AR protein contains a N-terminal transactivation domain, a DNA binding domain (DBD), a hinge region, and a C-terminal ligand binding domain (LBD). Upon agonist ligand stimulation, AR homodimerizes and translocates from the cytoplasm into the nucleus, where it can bind to specific DNA regions , to regulate the transcription of androgen responsive genes such as prostate-specific antigen , which can be constitutively activated without androgen stimulation and has increased expression under conditions of CRPC and abiraterone treatment  [Several mechanisms reported previously by ourselves and others may contribute to the reactivation of AR in CYP17 inhibitor-resistant PCa, including increased intratumoral steroid synthesis , 9, seleeatment) –20. Receeatment) –23. NeveAR-Q784* mutation identified in a CRPC patient who had been resistant to the treatment of ketoconazole, a CYP17 inhibitor. This point mutation converts glutamine to stop codon at amino acid 784 and creates a C-terminal truncated AR protein. As expected, AR-Q784* mutant produces an AR protein with partial truncation of LBD, which localizes in the nucleus and can bind to AREs without ligand stimulation. Surprisingly, AR-Q784* does not elicit transcriptional activity when overexpressed alone in AR negative cell lines. However, when co-expressed with AR-FL, this mutant AR can enhance AR-mediated transcriptional activity through possible heterodimerization with AR-FL in PCa cells. When AR-Q784* was overexpressed in an AR-positive PCa cell line, it enhanced the transcriptional activity of endogenous AR-FL particularly under low androgen conditions through stabilizing DNA binding of AR-FL and strengthening the AR recruitment of p300 coactivator, resulting in increased proliferation of PCa cells. These findings demonstrate that PCa cells under CYP17 inhibitor treatments may select for the expression of LBD-truncated ARs through a novel class of point mutations. In addition, our results suggest that expression of these AR mutants may function to enhance the activity of AR-FL under very low androgen conditions in CRPC treated with CYP17 inhibitors.In this study, we have characterized a novel nonsense AR, including progesterone-driven AR-T878A, are more frequent in CRPC and in CRPC resistant to more intensive ADTs [AR LBD mutations in CYP17A1 inhibitor treated tumors, we examined a set of CRPC samples obtained from CT-guided bone marrow biopsies from patients treated with high dose ketoconazole (a CYP17A1 inhibitor) and dutasteride (5α-reductase inhibitor), which were used to maximally suppress androgen synthesis [AR and wildtype AR are expressed in this sample, but whether they were coexpressed in the same tumor cell is unknown. PSA was also highly expressed (data not shown) but the expression of CYP17A1 was relatively low [AR gene produced a C-terminal truncated form of AR, which included only a fraction of the LBD  in wildtype AR to generate the AR-Q784* mutant. As seen in Figure AR-Q784* was translated to a protein that was smaller in size than wildtype AR (AR-FL), but larger than AR-V7. Unlike AR-FL, DHT had no stimulatory effect on AR-Q784* protein expression in PC-3 cells (an AR-negative PCa cell line), supporting that AR-Q784* is deficient in ligand binding ability  or activate the transcription of endogenous FKBP5 gene, a well-studied androgen-regulate gene, in either the absence or presence of androgen treatments  , 27 and AR at lysine 823 (AR-K823*), which produces a C-terminal truncated AR that retains both NLS and NES. In contrast to the constitutively nuclear localization of AR-Q784*, AR-K823* is largely excluded from nucleus and retained in cytoplasm regardless of hormone stimulation that is within LBD of AR (amino acid 742-817) . This NEonFigure  and 4B. PSA-enhancer) compared to AR-FL alone  that can be activated by progesterone, this mutant AR behaves similarly to the wildtype AR when stimulated by testosterone or DHT ligands [y Figure . InteresPSA mRNA transcription) was only modestly increased by expressing AR-Q784* upon high-level DHT (10nM) stimulation, the basal activity of AR was significantly enhanced over 2-fold  Figure . While Ad Figure . In contn Figure  and 6E, n Figure . Based oFKBP5-enhancer were examined. As seen in Figure We next sought to determine the molecular basis for the enhanced AR signaling in LN-AR-Q784* cells. To accomplish this, we examined the impact of the AR-Q784* mutant on chromatin binding of AR-FL and its coactivator recruitment. ChIP assays on full-length AR  and p300 (a well studied AR coactivator) were performed in doxycycline treated LN-AR-Q784* cells and their interactions with the AR mutations are relatively rare in untreated hormone-dependent PCa, they are more frequently detected in CRPC [AR mutations to allow the restoration of AR signaling in tumor cells. Recent studies on PCa cell lines and patient samples have revealed multiple AR point mutations, including treatment-induced F877L and T878A [AR alteration is the increased expression of AR splice variants, including AR-V7 or V12, which can constitutively activate transcription of AR-regulated genes despite the level of ligands [AR mutations that produces a partially LBD-truncated AR protein that structurally and functionally mimics certain LBD-truncated AR splice variants. Interestingly, unlike constitutively activated AR-V7 or V12, this mutant appeared to be a loss-of-function mutation when we studied its transcriptional activity in AR negative cells  mutations were previously identified as germ line mutations from patients with androgen insensitive syndrome, indicating this class of AR mutations are generally loss-of-function mutations (AR is located at X-chromosome) [AR mutations are generally rare in primary PCa. The expression of these mutations may not necessarily indicate loss of AR signaling since PCa cells can still harbor AR-FL allele due to the abnormality of chromosome numbers caused by chromosome instability. However, these mutations are likely passenger mutations due to the high-level androgen condition in primary PCa that can fully activate AR-FL. Importantly, the frequency of point mutations and gene amplifications of AR is substantially increased in CRPC samples [Similar LBD-truncation mutations of omosome) . This isomosome) , 34 alth samples , indicat samples . AlthougAR mutations.In summary, we highlight in this study the biological functions of a distinct class of AR variations, including nonsense or point-deletion mutations within LBD and certain splice variants (such as exon 6/7/8 escaping variants) that only lose a fraction of LBD domain (with disrupted NES), in CRPC or CRPC resistant to CYP17 inhibitor treatments. This class of mutants/variants are transcriptionally deficient but can enhance the activity of AR-FL stimulated by low level of androgen ligands possibly due to their constitutive DNA binding ability and dimerization with AR-FL. This function is consistent with many AR splice variants (such as V7 or V12) that can dimerize with AR-FL and enhance its activity in CRPC –23. HoweExpression vectors for pcDNA3.1(+), HA-tagged AR-FL (pcDNA3.1(+)-kozac-HA-HA vector backbone), and FLAG-tagged AR-FL (p3xFLAG-CMV-10 vector backbone) were kindly gifted from Drs. S. Chen and S. Balk at Beth Israel Deaconess Medical Center. AR-Q784* point mutation (C->T) and AR-K823* point mutation (A->T) were generated using QuickChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies Cat. # 210518) per the manufacturer's protocol. The primers for generating AR-Q784* are: forward, 5’-CT CATTCGGACACACTAGCTGTACATCCGGGAC-3’; reverse, 5’-GTCCCGGATGTACAGCTAGTGTGTCC GAATGAG-3’. The primers for generating AR-K823* are: forward, 5’-TTCATCAAAGAATTTTTGATTTTACAGCCCATCCACTGGAATAATG-3’; reverse, 5’-CATTATTCCAGTGGATGGGCTGTAAAATCAAAAATTCTTTGATGAA-3’. PLIX lentiviral vectors expressing AR-FL or AR-Q784* mutants were generated using the Gateway Technology with Clonase II (Invitrogen Cat #. 12535-029). Lenti-virus carrying PLIX-AR-FL or PLIX-AR-Q784* were assembled in HEK293T cells. LNCaP cells were then infected with lenti-virus in the presence of polybrene, followed by selection with puromycin.LNCaP cells were cultured in RPMI-1640 with 10% FBS . PC-3 cells were in F-12K medium with 10% FBS. COS-7 cells were in DMEM with 10% FBS. Stable LNCaP cell lines expressing empty vector, AR-FL, and AR-Q784* were maintained in tetracycline-free medium (RPMI-1640 supplemented with 10% tetracycline-screened FBS). For androgen stimulation assays, cells were grown to 50-60% confluence in hormone-depleted medium  for 2 days prior to treatment.For androgen stimulation assays, cells grown to 80-90% confluence in hormone-depleted medium for 24h were transiently transfected with pcDNA3.1(+) empty vector, FLAG-tagged AR-FL, HA-tagged AR-FL, FLAG-tagged AR-V7, or FLAG-tagged AR-Q784* using Lipofectamine 2000  for overnight, followed by 10nM DHT or other treatments for 24h.Firefly luciferase reporter construct together with a Renilla luciferase reporter construct for 24h prior to the treatments. Transfection was done in triplicates. Firefly luciferase and Renilla luciferase activity were measured using the Dual-luciferase Reporter Assay  and samples were normalized for Renilla activities.Cells were transfected with a For preparation of ChIP-qPCR, dispensed cells were formalin fixed, lysed, and sonicated to break the chromatin into 500–800 bp fragments, followed by immunoprecipitation. The qPCR analysis was carried out using the SYBR Green method on the QuantStudio 3 Real-time PCR system (Thermo Fisher Scientific). The primers of FKBP5 enhancer: forward, 5’-CTCTCCAACCTGCACTCCAT-3’; reverse, 5’-TAAA AGCACACAGGCGTGAA-3’. ChIP grade anti-AR  and anti-AR  were purchased from Santa Cruz, and anti-p300 was purchased from Abcam.Cells were harvested in triton lysis buffer containing protease inhibitors. The protein samples were then incubated with covalently immobilized anti-HA containing agarose beads (Sigma), followed by elution of IP-targets. The eluted proteins were then subjected to immunoblotting.PSA: forward, 5′-GATGAAACAGGCTGTGCCG-3′; reverse, 5′-CCTCACAGCTACCCACTGCA-3′; probe, 5′-FAM-CAGGAACA AAAGCGTGATCTTGCTGGG-3′, FKBP5 primers/probe mix was directly purchased from Thermal Fisher Scientific (assay ID: Hs01561006_m1 FKBP5). For immunoblotting, cell lysates were collected by boiling in 2%SDS for 15 minutes and anti-AR- , anti-AR- (for detection of AR-FL only) (Santa Cruz), anti-FLAG, anti-HA (Sigma), anti-LaminB1, or anti-GAPDH (Abcam) antibodies were used for blotting. Gels shown are representative of at least 3 independent experiments.RNA was extracted with TRIzol Reagent (Invitrogen Cat. # 15596018) based on manufacturer's protocol. The gene expression was measured using real-time RT-PCR analyses with Taqman one-step RT-PCR reagents on the QuantStudio 3 Real-time PCR system (Thermo Fisher Scientific) and results were normalized to co-amplified GAPDH. The primers and probes are listed below, COS-7 cells were transfected with AR-FL, AR-Q784*, or AR-K823*. NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific Cat. # 78833) were used to extract nuclear and cytoplasmic proteins following manufacturer's protocol. Immunoblot analysis was then performed as described above. For immunofluorescence staining, cells were fixed with methanol and then incubated with AR-441 antibody (Santa Cruz), followed by incubation with fluorescence labeled secondary antibodies . Micrographs were taken under EVOS auto fluorescence microscope (ThermoFisher) at the same microscope settings.TM Cell Analyzer using its Count & Viability Kit. Briefly, LNCaP stable cell lines were maintained in hormone-depleted RPMI medium (5% hormone-depleted FBS) and seeded in 6-well plates at 3 × 105 cells per well with same medium. After treatment, cells were collected and washed with PBS, followed by mixing with MuseTM Count & Viability Reagent. After 5 min incubation, cells were subjected to counting using Cell Analyzer.Cell proliferation assay was performed on an EMD Millipore Muset-test by comparing treatment versus vehicle control or otherwise as indicated. p-Value<0.05 (*) was considered to be statistically significant. Results for immunoblotting are representative of at least three experiments.Data in bar graphs represent mean±SD of at least 3 biological repeats. Statistical analysis was performed using Student's


Interferon regulatory factors (IRFs) can play a critical role in the regulation of many facets of innate and adaptive immune responses through transcriptional activation of type I interferons, other proinflammatory cytokines, and chemokines. However, their roles in transplantation immunity still remain to be elucidated. To evaluate the time course of mRNA expression of all 9 members of IRFs family of transcription factors during liver allograft acute rejection. Blood samples of 19 patients with autoimmune hepatitis receiving liver transplants were collected on days 1, 3, 5, and 7 post-transplantation. The patients were followed for 6 months after transplantation and divided into two groups of acute rejection (AR) (n=4) and non-acute rejection (non-AR) (n=15). vs. 1.91±0.27, respectively); expression of other IRFs family members was not significantly different between the two groups on days 3, 5, and 7 post-transplantation.All of the studied transcription factors were down-regulated in AR-group on days 3, 5, and 7 post-transplantation compared to non-AR group. The mean±SEM IRF5 on day 7 post-transplantation was significantly (p=0.005) lower in AR-group than in non-AR group (0.7±0.21 IRF5 may have an important role during the acute rejection of liver transplants. Autoimmune hepatitis (AIH) is a chronic inflammatory disease with unknown etiology. It has a mean incidence of 1.9 cases per 100,000 people per year and a prevalence of 16.9 cases per 100,000 people . In spitWhereas T cell responses are both necessary and sufficient for acute allograft rejection, most of researchers have been focused on adaptive immune responses in transplantation immunology . During Involvement of the innate immunity in AR is arising from the important role of Toll-like receptors (TLRs) as the first responders to danger signals . In receSignaling through TLRs can be categorized into two pathways: the MyD88 (myeloid differentiation primary-response protein 88) dependent pathway and TRIF (TIR domain containing adaptor inducing IFN-β) dependent pathway . Both ofIRFs family of transcription factors consists of nine members in humans: IRF1, IRF2, IRF3, IRF4 , IRF5, IRF6, IRF7, IRF8 , and IRF9  . Each IRet al, demonstrated that single nucleotide polymorphism in promoter region of IRF5 encoding gene, is correlated with acute rejection of liver allograft [Several gene expression profiling studies have so far reported the role of IRFs  in acute allograft rejection -20. Alsollograft . In addillograft , 24-26. This prospective study tried to clarify the role of IRFs family of transcription factors, in acute rejection after liver transplantation in AIH patients. We evaluated the time course of mRNA expression levels of IRFs in peripheral blood mononuclear cells (PBMCs) of patients with AIH who had acute and non-acute rejection of liver transplants.From May 2012 to March 2014, 20 Iranian adult female patients, who satisfied the international criteria for AIH , and recThe study protocol was approved by the Ethics Committee of both Shiraz University and Shiraz University of Medical Sciences, based on the study protocol conformed to the ethical guidelines of the 1975 declaration of Helsinki. Immunosuppressant RegimenAll recipients received the routine immunosuppression regimen consisted of tacrolimus or cyclosporine (CsA) with mycophenolate mofetil and steroids. Briefly, the drug dosage was adjusted to maintain target therapeutic blood levels of 200 ng/mL for CsA (5 mg/kg/day), or 10 ng/mL for tacrolimus. The patients with AR episodes received high dose (500 mg) methylprednisolone for three consecutive days.RNA Extraction and cDNA Synthesisst strand cDNA Synthesis Kit , according to manufacturer’s protocol.Total RNA was isolated from 1.5 mL fresh whole blood of recipients, immediately after sample collection, using QIAamp RNA blood mini kit  according to the manufacturer’s instructions. The purity and integrity of RNA was evaluated through measuring the optical density 260/280 ratio by spectrophotometric analysis. Then 500 ng of RNA samples with ratio above the 1.8 were used for cDNA synthesis by PrimeScript 1Quantitative Real-time PCRver 7.8 . Genomic DNA amplification was not appeared in our qPCR reactions, containing not reverse transcribed RNA as template. Real-time PCR primer sequences and conditions are presented in The mRNA expression levels of IRFs were determined by StepOnePlus real-time PCR instrument , using SYBR Premix Ex Taq II kit . The gene-specific primer sets were designed to span introns or cross exon/exon junctions, using AlleleID software Statistical Analysisver 16 . Data are presented as mean±standard error of the mean (SEM). A p value <0.05 was considered statistically significant.The statistical significance of differences in the measured gene expression levels between AR and non-AR groups was evaluated by Mann-Whitney U test, using SPSS Among 19 patients enrolled in this study, four with a mean age of 35 (range: 24–51) years experienced one AR episode during the six months of OLT; 15 patients with a mean age of 34.8 (range: 14–46) years did not experience AR during the study period. All AR episodes occurred within the first month post-transplantation. There were no significant difference in age, BMI, MELD score, total cold ischemic time, warm ischemic time, donor age, and sex, between AR and non-AR groups . No signvs. Non-AR groupExpression of IRFs in AR vs. 1.91±0.27, respectively). Expression levels of other IRFs family members were not significantly different between the two groups on days 3, 5, and 7 post-transplantation  lower on day 7 post-transplantation in AR-group than in non-AR group , as well as other types of liver autoimmune diseases were excluded. Therefore, it might be assumed that the comparison of IRFs expression levels between AR and non-AR groups, with the same underlying disease, represented the functional impact of these transcription factors in AR after OLT.Our results demonstrated that the mRNA expression of all nine members of this family decreased in AR compared to non-AR group on days 3, 5, and 7 post-transplantation, but only the down-regulation of IRF5 on day 7 post-transplantation was significant. It is well established that danger signals, released from donor organ as a result of ischemia-reperfusion injury, tissue damages, and hepatic phase of the transplant procedure, can activate Toll-like receptors, especially TLR4 , 30-32. et al [et al, showed that activation of TLR4 invokes nuclear translocation of IRF5 [Testro, et al , reporteet al , 36. Sevet al . A previ of IRF5 . Unlike  of IRF5 . AlthougIn conclusion, despite of our small study sample size and limitation in pre-transplantation sample collection, this study, for the first time, tried to represent evidence for the role of IRF5 in liver transplant AR. Further studies are needed to elucidate the functional impact of this transcription factor on liver transplant AR.


Patients infected with HIV have a significantly increased risk of developing non–Hodgkin lymphomas despite the widespread use of HAART.To investigate mTOR pathway activity in acquired immunodeficiency syndrome (AIDS) related diffuse large B-cell lymphoma AR-DLBCL, we used immunohistochemistry to examine the presence of the phosphorylated 70 ribosomal S6 protein-kinase (p70S6K), an extensively studied effector of mTOR Complex 1 (mTORC1) and the phosphorylated phosphatase and tensin homolog (pPTEN), a negative regulator of mTORC1 pathway.We evaluated tissue samples from 126 patients with AR-DLBCL. Among them, 98 samples were from tissue microarrays (TMAs) supplied by the Aids and Cancer Specimen Resource (ACSR), the remaining 28 samples were from cases diagnosed and treated at the University of California, San Diego (UCSD). The presence of p70S6K was evaluated with two antibodies directed against the combined epitopes Ser235/236 and Ser240/244, respectively; and additional monoclonal anti-bodies were used to identify pPTEN and phosphorylated proline-rich Akt substrate of 40kDa (pPRAS40). The degree of intensity and percentage of cells positive for p70S6K and pPTEN were assessed in all the samples. In addition, a subgroup of 28 patients from UCSD was studied to assess the presence of pPRAS40, an insulin-regulated activator of the mTORC1. The expression of each of these markers was correlated with clinical and histopathologic features.The majority of the patients evaluated were males (88%); only two cases (1.6%) were older than 65 years of age. We found high levels of both p70S6K-paired epitopes studied, 48% positivity against Ser235/236 (44% in ACSR and 64% in UCSD group), and 86% positivity against Ser240/244 (82% in ACSR and 100% in UCSD group). We observed more positive cells and stronger intensity with epitope Ser240/244 in comparison to Ser235/236 (p<0.0001). The degree of intensity and percentage of cells positive for pPTEN was positively correlated with p70S6K levels (p = 0.016 for 235/236 and p = 0.007 for 240/244). High levels of pPRAS40 were observed in the majority of the cases evaluated (64.3%), but no correlation was found with either pPTEN (p = 0.9) or p70S6K (p = 0.9) levels.AR-DLBCL frequently contain p70S6K, a main downstream effector of the mTOR pathway. The presence of p70S6K is positively correlated with pPTEN, an inactive form of PTEN, which makes mTORC1 activated. The presence of p70S6K was independent of HIV viral load or CD4 (+) counts. These results suggest that the mTOR pathway is active in the majority of AR-DLBCL, and p70S6K, particularly the Ser240/244 epitope immunohistochemistry is an excellent surrogate biomarker, which could be used to identify cases expected to be responsive to mTOR inhibitors. Infection with the Human Immunodeficiency Virus (HIV) makes patients more susceptible to viral induced neoplasms including acquired immunodeficiency syndrome (AIDS)-related diffuse large B-cell lymphoma (AR-DLBCL). Lymphomas in these patients often present at advanced stages, frequently with extra-nodal involvement, and have an aggressive clinical course . Co-infeIn recent years, the mammalian target of rapamycin (mTOR) has aroused much interest in cancer research. In the cancer setting, the most important control over mTOR activity is via the phosphoinositide-3-kinase/Akt—protein kinase B/mTOR (PI3K/Akt/mTOR) pathway , 6. Two There is evidence that mTOR is important in other AIDS-related cancers –10, and Available studies have evaluated the overall response rates with mTOR inhibitors at around 30% from DLBCL in immunocompetent patients , 14. FacIn order to investigate the extent to which the mTOR pathway is activated in AR-DLBCL, we evaluated the expression of p70S6K with two antibodies directed against the combined epitopes against Ser235/236 and Ser240/244 in 128 patients with AR-DLBCL. In addition, we also investigated the expression of pPTEN, a phosphorylated form of PTEN that activates mTOR pathway.http://acsr.ucsf.edu/). We analyzed the protein expression of p70S6K and pPTEN on formalin fixed paraffin embedded (FFPE) tissue  by immunohistochemistry (IHC). We then correlated these findings with histopathologic data, and if available, survival data, gender, CD4 count, whether or not subjects were taking antiretroviral medications, and cumulative viral load , but not pPTEN (r = 0.009 p = 0.97), or pPRAS40 . We further examined the potential associations between 70S6K, PTEN and PRAS40 phosphorylation and clinical features including nadir CD4 count, CD4 count at time of biopsy and response to chemotherapy.No significant associations were found. Finally, when a univariate analysis of associations between 70S6K epitope phosphorylation, viral load (VL), CD4 nadir, and CD4 count at time of biopsy with OS was performed, only CD4 count both at the time of biopsy and CD4 nadir were significantly associated with OS [ and (r = 0.73. P = 0.05)], respectively.In order to investigate if there is an alteration of gene expression in association with elevated mTOR signaling, gene expression profiles using the mRNA extracted from AR-DLBCL were performed. Due to technical difficulties of working with older formalin fixed samples, we were only able to extract mRNA from one patient tissue sample with activated mTOR and one patient tissue sample with minimal mTOR activity based on the IHC. While the examined samples are very limited, the results are very dramatic, namely, there is a uniform 3- to 6-fold increase in expression of the mTOR-related genes in mTOR activated case compared to minimally activated case . These rIn our study of the PI3K/Akt/mTOR pathway, effector proteins represented by 70S6K, PRAS40, and PTEN are shown to be phosphoylated in the majority of AR-DLBCL cases. At the protein level, in the case of downstream effectors 70S6K and PRAS40, this activation is manifested by the high number of cells and enhanced intensity observed of anti-p70S6K antibody for both paired epitopes (Ser235/236) and (Ser240/244) by IHC.In addition, the IHC demonstrated that the anti-p70S6K 240/244 antibody showed much higher levels than the anti-p70S6K-235/236 antibody indicating the 240/244 240/244 epitope is a superior surrogate marker for activation of this pathway in AR-DLBCL with 82–100% positivity in AR-DLBCL. In the case of upstream regulator PTEN, phosphorylation was also observed in the majority of AR-DLBCL, again consistent with the relief of the negative suppression on the PI3K/AKT/mTOR signaling pathway by PTEN .Our results are in agreement with those of El-Salem et al. who identified p70S6K activation in the majority of 24 cases of AR-DLBCL, although they did not report the exact percentage of positive cases  who repoIn agreement with the phosphorylation of 70S6K in the majority of the AR-DLBCL cases, the phosphorylation of PTEN, a tumor suppressor and the major negative regulator of the PI3K/AKT/mTOR pathway, was observed in 68–77% of AR-DLBCLs, substantiating our findings that the negative suppression of mTOR in AR-DLBCL was relieved by PTEN. Of interest, non-AR-DLBCL also has similar PTEN findings. However, in non-AR-DLBCL, Pfeifer et al. report 47% of GCB DLBCLs stained for PTEN in comparison to 87% in non-GCB DLBCLs leading to a proposal of PTEN loss that defines a PI3K/AKT dependent pathway in GCB DLBCL . In our The AR-DLBCLs from UCSD patients were associated with extensive clinical data, which were unfortunately not available from the ACSR samples. In AR-DBCLs from UCSD, we did not observe a significant correlation between cumulative viral load and 70S6K phosphorylation. Similarly, no association was observed between 70S6K phosphorylation and CD4 count, either at time of biopsy or at nadir. While there could be no true association between 70S6K phosphorylation and viral load as well as CD4 count, the small sample size could be the real reason. In this small cohort, OS was only associated with CD4 count at time of biopsy and CD4 nadir, consistent with the published literature .The majority of studies of the mTOR signal pathway in non-Hodgkin lymphomas have been performed on cell lines , 22. In In conclusion, we show that AR-DLBCL exhibits high levels of mTOR expression. Immunohistochemistry of the surrogate marker, p70S6K and pPTEN, could potentially be used to predict activation of mTOR, and identify cases potentially responsive to rapamycin and its analogs as adjuvant therapy.S1 Table(DOCX)Click here for additional data file.S1 File(ZIP)Click here for additional data file.


Androgen receptor (AR) has been shown to have prognostic implication on breast cancer (BC). Data on the biological features of African BCs are poor. We decided for the first time to compare AR expression of Tanzanian and Italian BC patients.Of the 69 consecutive patients seen at the Bugando Medical Center  from 2003 to 2010, who underwent resection of primary BC evaluable for estrogen receptor, progesterone receptor (PgR), and HER2 only 65 were evaluable for AR by immunohistochemistry. Histopathological assessment and biomolecular determinations were performed at the Cancer Institute of Romagna . Caucasian BC patients were selected from an electronic database and matched (1:2 ratio) for year of diagnosis and age at diagnosis.p < 0.0001) and staining intensity (p = 0.0003). The proportion of AR negative patients was likewise higher among Tanzanian patients as regards both ≥1% and ≥10% cutoffs. AR-positive BCs were higher in luminal A and B tumors and decreased in triple-negative (TN) and HER2-enriched tumors in Tanzanian population.The median age of patients at diagnosis was 51 (range 29–83) years for Tanzanian and 53 (range 26–86) years for Italian patients. Tanzanian patients harbored tumors with lower AR expression than Italian patients according to the median percentage of immunopositive tumor cells (30% versus 80%, AR loss could represent an unfavorable prognostic marker in the African population. The high frequency of TN tumors with high AR expression could open new perspectives of therapy for population in this low income country. The mortality for breast cancer (BC) in Tanzania is the second cause of cancer death after cancer of the uterine cervix, and the major part of cancer being diagnosed very late at advanced stage .Breast cancer incidence estimated age standardized rates in sub-Saharan Africa range from 15 to 53 per 100,000 women, so lower than Western countries . The canThe prognosis and treatment of BC patients depend on numerous clinical, pathological, and biological factors. Their evaluation is important to classify BC into categories, that have different impact on treatment choice and prognosis . The current treatment choice for BC in developed countries is influenced by numerous factors, including the risk of relapse, breast conservation, and impact on fertility. Surgery and radiotherapy play an important role for the treatment of early BC. Tamoxifen, with or without ovarian suppression, is the standard of care for women in premenopausal status with ER+ BC, while aromatase inhibitors are preferred for postmenopausal women. Luminal B and TN BCs are commonly treated with anthracyclines and taxanes, since chemotherapy lacks of a single standard of care. In addition to chemotherapy, for HER2-enriched BC patients, 1-year adjuvant treatment with Trastuzumab is recommended .However, due to the poorer pathology skills and infrastructures in most sub-Saharan African countries, hormonal receptors, HER2, and Ki67 are not routinely assessed, and BC patients often undergo hormone treatment even though the receptor status is unknown .Then, proper infrastructures and improved professional human skills and technical facilities are necessary to permit the biological and pathological BC characterization for each patient , 5.Some authors have reported that African-American premenopausal women have a higher likelihood of developing BC with the “TN” phenotype .Androgen receptor (AR) in BC is commonly expressed in the 60–80% of luminal tumors, 50–60% of HER2-enriched, and in the 20–40% of TN tumors , 8.in situ (DCIS) of the breast treated with surgery alone -positive and HER2-negative BC . Severalry alone  and in aIn our previous work, we observed that BC in Tanzanian patients at time of diagnosis more frequently presented a higher histological grade (mainly grade 3), more advanced clinical stage (III or IV), ER negativity and higher proliferation index than those in Caucasian patients . We concIn this work, we want to compare AR expression in these two populations of BC patients in the different tumor subtypes. The availability of anti-AR compounds such as bicalutamide, enzalutamide, or apalutamide could open up new avenues for the treatment of AR-positive tumors –14.In this study, we compared the biological characteristics of Tanzanian and Italian BC patients matched (ratio 1:2) for date and age at diagnosis. The Medical Scientific Committee of IRST IRCCS, the Ethical Committees of Area Vasta Romagna  and BMC (Tanzania), and the National Institute for Medical Research (Tanzania) approved the study. The informed written consent from the participants was obtained.A total of 69 consecutive patients who underwent biopsy or surgical resection of primary BC from 2003 to 2010 at the BMC  were enrolled in this study. Formalin-fixed paraffin-embedded tissues were analyzed for AR expression in the Biosciences Laboratory of the Cancer Institute of Romagna (IRST IRCCS) in Meldola, Italy.Breast cancers from Caucasian patients were randomly extracted from an electronic database (Log80) of the Pathology Unit of Morgagni-Pierantoni Hospital  and matched with Tanzanian patients for year of diagnosis and age at diagnosis (maximum difference of 2 years). The former stratification factor was chosen to avoid biological material alteration due to the long enrollment period, and the latter was chosen because age can affect the analysis of biomarkers in BC.The clinical and pathological assessments of these cases were performed at the Oncology Unit and Pathology Laboratory of BMC, while hormone receptor status [ER and progesterone receptor (PgR)], HER2 expression, and proliferative activity (Ki67) assessments were previously done at the Biosciences Laboratory of IRST IRCCS. In this work, only AR expression was analyzed for the entire case series in our Institute.Four-micrometer sections of neutral buffered formalin-fixed, paraffin-embedded tissue were mounted on positive-charged slides .Androgen receptor determinations were performed according to European Quality Assurance guidelines.Immunostaining for AR was performed using the Ventana Benchmark XT staining system , with Optiview DAB Detection Kit . AR  antibody was ready to use, prediluted by supplier, and sections were incubated for 16 min. The sections were automatically counterstained with hematoxylin II .H score defined as the product of the percentage of the immunopositive tumor cells and the staining intensity.Androgen receptor-positive samples were classified by using two different cutoffs ≥1% and ≥10% of nuclear immunopositive tumor cells in the nucleus on the total of the tumor cells. Staining intensity  was also analyzed to calculate Moreover, positive and negative breast tissues were used as intra- and inter-assay controls for AR expression. All samples were evaluated by two independent observers. A disagreement of more than 10% of positive cells was resolved by consensus after joint review using a multihead microscope.All the data were summarized using descriptive statistics .rs coefficient) was used to investigate the relation between AR and Ki67 status. Statistical tests were two sided and were significant for p values < 0.05. Analyses were performed using SAS software version 9.4 .The chi-square test or Fisher’s exact test for categorical variables and the Wilcoxon–Mann–Whitney test for continuous variables were performed to identify significant differences between the Italian and Tanzanian populations. Spearman’s rank correlation test (p = 0.258). Moreover, the invasive ductal cancer represented the 92.3% of the African BC population and the 81.5% of the Caucasian one. Tanzanian patients presented more frequently disease at advanced stage (III–IV) than Italian patients (p < 0.004) (Table 199 patients were included in the study: 69 patients from Tanzania (100% African ethnicity) and 130 from Italy (100% Caucasian ethnicity). Of 69 Tanzanian BC cases of the overall series only 65 had tumor tissue to assess AR status and were matched with 130 Italian BC patients for age and date of diagnosis. The clinical–pathological features of the two case series are reported in Table p < 0.0001)  in Tanzanian and 240 (0–300) in Caucasian patients (p = 0.109) (Table p = 0.0003) (Table The median AR expression (% of immunopositive tumor cells in the nucleus) in Tanzanian BC patients was 30 range 0–100) and in Caucasian population was 80 (range 0–100) , for the former the rs is −0.21 (p = 0.209) and the latter showed an rs of −0.23 with a p-value of 0.010.In addition, we evaluated the correlation between AR and Ki67 status in primary tumors. In the overall series of African and Caucasian tumors taken together, the Information on BC biomarkers is poor in the majority of low-resource countries, such as Sub-Saharan Africa. It is worthy of note that pathology capacity and infrastructures are insufficient in most parts of sub-Saharan Africa , 5, 15. These highly aggressive biological patterns, with very advanced stage at diagnosis, could be considered the principal reasons for the high BC mortality rate in this African population. Then, the search for new biomarkers that could be used in the clinical practice is still an open issue and even more the identification of biomarkers to optimize the treatment choice.Androgens have been thought to play an important role in BC. Even if we know that our results are preliminary due to low number of cases analyzed, in our knowledge this is the first study that compares the pathological and the biological features and AR expression in invasive BC in African (Tanzanian) and Caucasian  case series. Another study evaluated AR expression in Ghanaian BC patients. They found a lower percentage (24%) of AR-positive tumors (defined by ≥10% cutoff) among TN BCs .H score, and staining intensity. We are in agreement with Thike and colleagues that reported that the lower AR expression reflects the higher aggressiveness of tumors, but their study was performed in a different ethnicity, such as Asian population  leading to massive proliferation and invasiveness of target cells .Moreover, an interplay between AR and ER has been known, and it is exerted at the level of estrogen responsive elements . The croMigliaccio and colleagues demonstrated that a non-genomic interplay between AR and ER can occur at protein level involving Src tyrosine kinase and epidermal growth factor receptor , 21.Several coregulators balance the activity of these two hormone receptors and their interactions in different clinical settings. Some therapeutic approaches can be based on blocking this cross talk .In Caucasian patients, AR was seen to be more expressed in luminal tumors than TN tumors, and its presence seems to be related to a better prognosis in ER-positive tumors –26. The In the Tanzanian clinical practice, all patients underwent adjuvant hormonal therapy without testing the receptors. It means that only the fraction of ER-positive patients would benefit from this type of treatment. On the other hand, the majority of the patients (about 70%) were exposed to hormonal therapy unnecessarily, with subsequent side effects and additional costs in a low income country. The availability of new anti-AR compounds could lead to treat the patients with reduced recurrence, mortality, and costs , 5.This area needs further investigation as the data on the differences of gene expression profiles in BC patients of various ethnicities are still controversial , 28. ThiThe new availability of AR inhibitors such as bicalutamide, enzalutamide, and apalutamide approved for prostate cancer, suggests the possibility of their use also in AR-positive BC patients, even if AR seems to have different functions depending on BC subtypes . It seems that in ER negative BCs AR expression does not have a clear prognostic effect , but it It is recommended to introduce in Tanzania routine testing for these markers before initiation of hormonal therapy and also to consider an anti-AR therapeutic approach. Further analyses are ongoing to evaluate the role of other biomarkers in Tanzanian BCs.The study was approved by the Medical Scientific Committee of IRST IRCCS, the Ethical Committee of Area Vasta Romagna  and BMC (Tanzania), and the National Institute for Medical Research (Tanzania).SB and DA designed the study. AR, NM, JK, AP, LF, AF and RM were responsible for patients’ recruitment. SR and MT performed the experiments. PS performed data management. ES performed the statistical analyses. MP and SB performed the immunohistochemical evaluation of all the samples. SB and GB interpreted the results and drafted the manuscript. MB revised the text. All the authors read and approved the present version of the manuscript for submission.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Purpose/Objective. To investigate the effect of Actaea racemosa (AR) extract on in vitro osteogenic differentiation of rat bone marrow mesenchymal stem cells (BMSCs) via the ER/NO/cGMP signaling pathway. Methods/Materials. Rat BMSCs were treated with osteogenic differentiation-inducing medium containing AR; estrogen receptor antagonist, ICI 182,780 (10−6 mol/L); and nitric oxide synthase inhibitor, L-nitro arginine methyl ester . Markers of osteogenic differentiation  and the levels of key signaling molecules  were assessed. Results. AR (10−1–10−6 g/L) increased ALP activity in a dose-dependent manner, and the highest ALP, osteocalcin, and osteoprotegerin activities were achieved at an AR concentration of 10−4 g/L. Therefore, the concentration of 10−4 g/L was used for promoting osteogenic differentiation of BMSCs in subsequent analyses. At this concentration, AR increased the levels of NO and cGMP, and such effects could be blocked by the estrogen receptor antagonist  and nitric oxide synthase inhibitor (L-NAME). Conclusion. AR induced osteogenic differentiation of rat BMSCs through the ER/NO/cGMP signaling pathway. This finding provides the theoretical foundation for the mechanism of AR in the treatment of postmenopausal osteoporosis. Postmenopausal osteoporosis (PMOP) is a prevalent disease that affects 50% of women around the age of 60 mainly due to estrogen deficiency. Estrogen replacement therapy is effective in preventing and treating PMOP, but its risks outweigh its benefits . TherefoActaea racemosa (AR) extract has long been used to treat perimenopausal syndrome because it does not contain estrogen but has effects to alleviate menopausal symptoms, particularly hot flushes, night sweats, and correlated sleep disturbances [ In vitro experiments showed that AR increased the levels of osteoprotegerin (OPG), alkaline phosphatase (ALP), and osteocalcin (OC) secreted by osteoblasts and promoted the formation of mineralized bone nodules as well as the expression of Runx2 and OC in MC3T3-E1 precursor cells [−6 mol/L). Interestingly, bone tissue is rich in estrogen-β-receptors (ER-β) in contrast to ER-α-rich tissues such as breast or endometrium. Therefore, this was indicating that AR has a positive effect on the bone partially through ER signaling [urbances , 3. In aurbances –11. Clinurbances , 13. Expurbances –16. Howeor cells , 18. Morignaling , 18.Recent studies have shown that the nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) signaling pathway is closely related to bone metabolism. NO inhibits bone resorption by osteoclasts and promotes differentiation of osteoblasts . In addin = 8) weighing around 120 g were obtained from the Experimental Animal Center, Tongji Medical College, Huazhong University of Science and Technology (HUST), under the Animal Quality Certificate Number SCXK 2010-0009. Notably, all experiments were performed in accordance with HUST's ethical guidelines for animal care as well as the guidelines set by the World Health Organization, and the experimental protocols were all approved by the animal care committee of HUST.Specific pathogen free- (SPF-) grade adult male Sprague Dawley (SD) rats  to supplement with varying concentrations of 10−1–10−6 g/L. The following were obtained: DMEM/F12 medium with phenol red, fetal bovine serum ; phosphate buffer solution (PBS), 0.25% pancreatin, and BCA Protein Assay Kit ; ER antagonist ; ALP Assay Kit, OC Assay Kit, OPG Assay Kit, NO Assay Kit, NOS Assay Kit, Calcium Assay Kit, rat cGMP ELISA Kit, and nitric oxide synthase inhibitor L-NAME (Nanjing Jiancheng Bioengineering Institute); anti-rat CD29-PE, anti-rat CD90-FITC, anti-rat CD45-APC, and anti-rat CD11b-PE-Cy7 ; Trizol reagent ; trichloromethane, anhydrous ethanol, and isopropanol ; HyPure™ Molecular Biology Grade Water ; Revert Aid First Strand cDNA Synthesis Kit ; Fast Start Universal SYBR Green Master (Rox) . All primers were synthesized by Invitrogen Biotechnology Co., Ltd.  was dissolving with 70% ethanol/water (1 mL) and then using an osteogenic induction medium  was centrifuged for 5 min at 1500 rpm, and the supernatant was discarded. Cells were blocked in BSA solution at 4°C for 1 h and centrifuged as before. The cells were stained with anti-rat CD90-FITC, anti-rat CD29-PE, anti-rat CD45-APC, and anti-rat CD11b-PE-Cy7 in the dark for 20 min. The cells were then washed with PBS to remove unbound antibodies and immediately analyzed on a flow cytometer to determine antigen expression and cell purity.The third subculture of rat BMSCs , and the cells were incubated for 8 days. The osteogenic induction medium supplemented with varying concentrations of AR was changed every two days. Cells were harvested on day 8 and the ALP, OC, and OPG activities were determined . By com−4 g/L); AR + ICI 182,780 (10−6 mol/L); AR + L-NAME; ICI 182,780 (6 × 10−3 mol/L) [When cells were approximately 80% confluent, the normal culture medium was replaced with the osteogenic induction medium, and cells in the AR (103 mol/L) , calciumμL) was evenly mixed with detection reagent, and color development was measured as absorbance at 520 nm using a microplate reader. The total secreted protein content was determined using a BCA Protein Assay Kit. The secreted ALP activity (expressed in U/g prot), OC activity (expressed in ng/mL), and OPG activity (expressed in ng/L) in each group [Cells were collected on day 8 after treatment, lysed as previously described, and centrifuged . The supch group  were calμL) was evenly mixed with detection reagent and color development was measured as absorbance at 610 nm using a microplate reader. The total secreted protein content was determined using a BCA Protein Assay Kit. The Ca2+ content in each group, expressed in mmol/g prot, was calculated according to the absorbance, concentration of standard solution, and protein concentration of the sample to be tested in each group.Cells were collected on day 12 after treatment, lysed as previously described, and centrifuged. The supernatant  and was calculated based on corresponding absorbance and reference standard concentrations.The NO content in the supernatant of cells was determined using the nitric oxide assay kit, according to the manufacturer's instructions. The NO content in the supernatant in each group was calculated according to synthase activity  was used and the expression levels of ALP, eNOS, and iNOS were determined. The CT values (ΔΔCT method) were calculated and analyzed as previously described [Treated cells were collected and total RNA was extracted using TRIzol reagent. The Revert Aid First Strand cDNA Synthesis Kit was used to reverse-transcribe the RNA into corresponding cDNA. Using cDNA as the template, 2x qPCR Mix and 7.5 escribed . The priP < 0.05 was considered statistically significant.Statistical analyses were performed by analysis of variance (ANOVA) using SPSS software . All test data were expressed as mean ± standard deviation, and Primary cells were cultured for 24 h followed by changing the culture medium. After 9–12 days of incubation, cells grew to approximately 80% confluence in a monolayer with cells arranged in specific directions. During subculturing, cells grew considerably faster and reached 80–90% confluence within 5-6 days. Cells at the third subculture had a long spindle-shaped morphology and were densely arranged in a swirling pattern . Flow cy−1–10−6 g/L AR. As shown in P < 0.001). AR did not stimulate highest activities of ALP, OC, and OPG at concentration of 10−1 g/L (highest doses of AR) and 10−6 g/L (lowest doses of AR) but at 10−4 g/L, which showed a significantly increased stimulating potentiate bone forming activity. Therefore, the concentration of 10−4 g/L was used for promoting osteogenic differentiation of BMSCs in subsequent analyses.BMSCs were treated with 10P < 0.001, Figures β-actin expression were 16.065 ± 0.213, 16.167 ± 0.204, 16.158 ± 0.209, 16.081 ± 0.201, 16.146 ± 0.215, and 16.131 ± 0.212 in the control group, AR group, AR + ICI 182,780 group, AR + L-NAME group, ICI 182,780 group, and L-NAME group, respectively. They were not significantly different from each other ; therefore, it could serve as housekeeping gene. AR increased the ALP mRNA expression 2.05 times as compared to that in the control group . This effect was blocked by ICI 182,780 and L-NAME treatment. Similarly, cells treated with either ICI 182,780 or L-NAME alone exhibited no difference in calcium ion deposit as compared to that in the control group   concentration of AR was also found to be the largest enhanced bone nodules formation [The safety and efficacy of AR in the treatment of perimenopausal syndrome have been confirmed , 3, but ormation .β-estradiol promotes the functions of osteoblasts through the ER receptor signaling pathway [eNOS can be expressed at low levels in osteoblasts, osteoclasts, and marrow stroma cells. Studies show that eNOS is an indispensable molecule in the process, whereby 17 pathway . iNOS is pathway . eNOS pr pathway . In this pathway . AR may cGMP plays an important role in the proliferation and differentiation of osteoblasts. Increased NO levels activate soluble guanylate cyclase to increase the cGMP content and accelerate the differentiation and mineralization of osteoblasts through the action of cGMP –37. Here−9 mol/L increases the concentration of NO and cGMP to the highest levels, and these effects could be blocked by the estrogen receptor antagonist  and NOS inhibitor (L-NAME). This suggests that AR plays a very important role in promoting the differentiation of BMSCs into osteoblasts through the ER/NO/cGMP signaling pathway. While these findings provide a secure foundation, additional work is warranted in order to fully understand the role of this herbal medicine in osteoporotic differentiation of BMSCs.This study contributes important expansion of recent experiments on possible clinical application of AR for preventing PMOP. These experiments highlight the use of AR to induce the passage and differentiation of BMSCs isolated from osteoporotic rat into osteoblast-like cells. AR at 10


Androgen and its receptor (AR) play an important role in maintaining spermatogenesis and male fertility. The nonclassical androgen signaling pathway is proposed to be mediated by an AR in plasma membrane in Sertoli cells. Our previous studies showed that testosterone induces cytoplasmic AR translocation to plasma membrane by binding with caveolin-1. This study was conducted to the underlying molecular mechanism mediating AR trafficking. Data from mass spectrometry using membrane coimmunoprecipitation sample by anti-AR antibody indicated VAPA is a candidate protein. Knockdown of VAPA by shRNA decreased the amount of AR localized to membrane and nuclear fraction and prevented AR trafficking after being exposed to testosterone. Further studies indicated AR trafficking in Sertoli cells might be mediated by VAPA via association with vesicle transport protein OSBP. This study can enrich the mechanism of the androgen actions and will be helpful for further clarifying the nonclassical signaling pathway of androgens in Sertoli cells. AR knockout mice exhibit spermatogenesis arrest at the diplotene stage [AR knockout mice. The selected AR knockout in germ cells does not affect the process of the spermatogenesis [Androgen and its receptor (AR) play an important role in maintaining spermatogenesis and male fertility. Evidences showed that the actions of androgen in Sertoli cells were essential for the proceeding of spermatogenesis. Sertoli cell-selected ne stage –3, similogenesis . These dAR associates with testosterone, and the complex translocates to nucleus, binds with the androgen response element (ARE) in the chromatin, and then induces gene transcriptional repression or activation. This is the classical pathway of androgen, taking several hours since it requires new protein synthesis and secretion. Compared with the classical pathway, a faster action of androgen called nonclassical signaling pathway was found. It only takes several seconds to minutes to response since it does not relate to DNA amplification or protein synthesis. And evidences suggested this fast action is mediated by a membrane receptor of androgens. Our previous data showed AR localized in the membrane of murine testicular TM4 cells, and testosterone enhanced the membrane association, AR localizes to plasma membrane by binding to Caveolin-1 , 6. HoweIn this study, we used coimmunoprecipitation (co-IP) and employed mass spectrograph (MS) to find the candidate protein involved in AR trafficking. The screened candidate proteins will be knock down using shRNA constructs to test the role in AR trafficking. We will also conduct experiments to study the molecular mechanism mediating AR trafficking by the candidate protein. This study will enrich the mechanism of the action of androgens and provide new sights into testosterone signaling pathway in Sertoli cells mediating spermatogenesis.Unless otherwise indicated, all the chemical reagents were obtained from Sigma-Aldrich (USA), all of the cell culture reagents were bought from Life technologies (USA). The shRNA construct was provided by Shanghai Genechem Co. Ltd (China).μg/ml penicillin and 100 μg/ml streptomycin at 37°C. Cells plated in 10 cm culture dishes were transfected with shRNA or negative control using Lipofectamine 3000 (Invitrogen). Cells were changed to medium supplemented with stripped serum when reaching 80%–85% confluence. Eighteen hours later, the medium was changed to serum-free medium containing 0.1% BSA and incubated for 2 hours before exposure to 10 nM testosterone  for 30 minutes unless otherwise indicated.Murine Sertoli cell line TM4 was purchased from the American Type Culture Collection . Cells were cultured in DMEM containing 10% FBS and 100 μg of protein was subjected to immunoprecipitation using 2.5 μg of rabbit polyclonal antibody, anti-AR N-20 (Santa Cruz Biotechnology) and 2.5 μg of rabbit polyclonal antibody, anti-AR . After separation of immunoprecipitated proteins using magnetic protein A/G Dynabeads (Invitrogen), pellets were washed by lysis buffer  three times, 5 min each. Proteins were disassociated from the magnetic beads by incubating with 0.1 M glycine (pH 2.2–2.7) for 10 min. A part of co-IP samples was sent to FitGene Biotechnology Co. Ltd.  for mass spectrometry (MS) detection and bioinformatic analysis, and the other was for Western blot analysis.Protein subcellular fractionation isolation was following the instruction provided in the kit as previously described . 500–100μg of protein or equal volume of co-IP samples. The samples were subjected to SDS-PAGE on 10% tricine-glycine gels and transferred onto polyvinyl difluoride membranes (Millipore). After blocking nonspecific protein-binding sites by incubation for 1 h at room temperature in TBST  containing 5% nonfat dry milk, membranes were incubated overnight with primary antibody  and increased in the cytoplasmic fraction under testosterone treatment (P < 0.01). These data indicated that VAPA may be involved in the AR membrane trafficking.Vesicle-associated membrane protein-associated protein A (VAPA) presents in the plasma membrane and intracellular vesicles. This protein is reported in function in vesicle trafficking, membrane fusion, protein complex assembly, and cell motility. Membrane and cytoplasmic fractions were isolated from TM4 cell with or without testosterone administration. As shown in P < 0.01). Cells transfected with negative control or VAPA shRNA were exposed to 10 nM testosterone for 30 min. Compared with the negative control, blots and data analysis showed a decrease in the membrane  and nuclear  localization at basal condition ; and the nuclear trafficking was also inhibited .shRNA of VAPA was designed and synthesized to decrease the expression of VAPA in TM4 cell. Western blot and RT-PCR results in  Figures . After eP < 0.01) but increased in the cytoplasmic fraction . The data analysis also showed that the association of VAPA and OSBP was increased in cytoplasmic fraction  but unchanged in membrane samples.Experiments were conducted to study the molecular mechanism of AR trafficking mediated by VAPA. It was reported that VAPA interacts with OSBP (Oxysterol-binding protein) . The expSpermatogenesis is a complex and highly ordered process regulated by a series of gene products. Lots of clinical and experimental studies suggested that androgen and its receptor are critical for spermatogenesis and male fertility. Androgen controls spermatogenesis mainly by associating with the AR localized in Sertoli cells, and the role of androgen in Sertoli cells includes maintaining blood-testis barrier, meiosis, Sertoli-spermatid adhesion, and sperm release.AR is capable of transmitting testosterone signals by at least 2 mechanisms, the classical and nonclassical pathways. In the classical signaling pathway, testosterone that diffuses through the cell membrane interacts with AR in the cytoplasm and then translocates to nucleus, binds AREs in gene regulatory regions, and recruits coactivator or corepressor proteins to regulate gene expression .In the nonclassical pathway, stimulation of Sertoli cells with levels of testosterone 10–250 nM) that are similar to or lower than that found in the testis causes a population of AR to localize near the plasma membrane. Studies using Western blot and immunofluorescence confirmed the localization of AR in membrane –250 nM t. In a prIn order to figure out the molecular mechanism of AR trafficking to membrane fraction, we identified the candidate proteins involved in this action using MS and bioinformatic analysis. 10 candidate proteins were identified; we conducted experiments to confirm the data and study the molecular mechanism mediating AR trafficking by these proteins.VAPA is a vesicle membrane-associated protein, evolution conserved in species and participates the formation of lipid bilayer of the tight junction between endoplasmic reticulum and of other organelles. VAPA is a type IV membrane protein, localizes in the plasma membrane and intracellular vesicles . It may 4P) [VAPA has been shown to interact with the OSBP to modify export from the endoplasmic reticulum . It is r4P) . The bloIn this study, we identified proteins involved in cytoplasmic AR translocation to plasma membrane under the treatment of testosterone at physiological concentration, and the data showed VAPA plays an important role in this process. Co-IP and Western blot results verified the MS analysis; further studies showed a decrease in AR membrane localization and trafficking in cells transfected with VAPA shRNA. AR trafficking in Sertoli cells might be mediated by VAPA via association with vesicle transport protein OSBP. This study is able to further enrich the molecular mechanism of the androgen actions in Sertoli cells and provide new insights into the nonclassical signaling pathway of testosterone, which mediate spermatogenesis.