{"paper_id":"d038428a-c11a-4b4b-98d9-30cd360644d1","body_text":"Adiponectin is one of the most important\nmembers of adipokine family which is widely\nsynthesized and secreted by fatty tissue. Various\nroles have been identified for adiponectin such\nas regulation of glucose level and lipids homeostasis.\nFurthermore, adiponectin plays a pivotal\nrole in reproductive system ( 1 ,  2 ). Adiponectin\nis abundantly present in the blood stream and\nits concentration in human plasma is 5-30 μg/\nml which comprises 0.01% of all proteins in the\nplasma ( 3 ).\nDecreasing adiponectin of plasma is indicative\nof obesity and diabetes ( 4 ). Also, various studies\nhave demonstrated that decreasing adiponectin\nplasma level is linked to increasing the risk of\nseveral type of cancer, including breast cancer ( 5 ),\ncolorectal ( 6 ), prostate ( 7 ), and digestive system ( 8 ). Adiponectin binds to receptors, known as\nAdipo R1 and Adipo R2 ( 9 ). These receptors\ncontain seven transmembrane domains but differ\nfrom G-protein coupled receptors structurally\nand functionally. The tendency of adiponectin\nreceptors to bind to adiponectin isoforms as\nwell as tissue distribution of these receptors are\ndifferent ( 10 ).\nIn mice, Adipo R1 exists in different organs\nsuch as skeletal muscle, lung, and spleen;\nwhereas Adipo R2 is mainly expressed in liver\n( 11 ). In human, Adipo R1 and Adipo R2 are expressed\nin islets of Langerhans, macrophages,\nadipocytes, and vascular smooth muscles ( 12 -\n 14 ).\nVarious data have indicated that adiponectin\nis influential in female fertility and plays an\nimportant role in female reproductive system.\nStudy has indicated that serum adiponectin level\ndecreases in women with endometriosis ( 15 )\nand endometrial cancer ( 16 ). Also, adiponectin\nlevel in peritoneal fluid of endometriosis patients\ndecreased dramatically in advanced endometriosis\n( 17 ).\nIn histopathological studies of endometriosis\ntissues, stromal cells and glands are abundantly\npresent, but changes of endometrial stromal\ncells are much more than those of endometriosis\nidentifying glands and there is the possibility\nof the presence of gland-free stromal cells in\nendometriosis tissue ( 18 ). HESCs play pivotal\nrole in female reproductive biology and there is\nno report on the effect of adiponectin on these\ncells. The aim of the present study was to examine\nthe effect of adiponectin on human endometrial\nstromal cells and  in vitro  mRNA expression\nof adiponectin receptors.\n\nIn this experimental study, endometrial tissues\nwere taken from women aged 25-35 who\nhad no record of hormonal treatment for three\nmonths before surgery and had undergone hysterectomy\nsurgery or biopsy diagnosis for infertility\nmanagement and reasons other than endometrial\nmalignancies such as myoma. Eight\nsamples of normal endometrium in the secretory\nphase were taken. The Ethics Committee\nof Kermanshah University of Medical Sciences\nand Tehran Science and Research Branch of\nIslamic Azad University accepted the work on\nhuman endometrial tissue in this study and all\npatients signed informed consents.\nStromal cells were separated from endometrial\ntissue according to previous work ( 19 ,  20 ).\nEach endometrial sample was prepared in sterile\ncondition and was washed with PBS solution\ncontaining 1% antibiotic/antimycotic, and\nthen was chopped mechanically. The sample\nwas incubated with collagenase type I solution\n(2 mg/ml in DMEM/F12) (Sigma, Germany) for\n60-90 minutes. The cell suspension was passed\nthrough 70 and 40 μm cell strainers (BD falcon,\nUSA) respectively, centrifuged for 15 minutes\n(2500 rpm) and DMEM/F12 (Gibco, Germany)\nwas added to the cell pellet. Then the suspension\nwas layered on ficoll (Amersham, Sweden)\nand was centrifuged for 30 minutes (1500\nrpm). The stromal cells were collected and were\nwashed with PBS and were cultured in DMEM/\nF12 containing 10% fetal bovine serum (FBS)\n(Gibco, Belgium), 0.1 mg/ml streptomycin, and\n100UI/ml penicillin. The cultures were incubated\nin a humidified atmosphere of 95% air and\n5% CO 2  at 37˚C. After seven days, cell density\nreached confluency and 1×10 5  cells were transferred\nto each well of 24-well culture plate.\nTo add adiponectin (high molecular weight,\nR&D System Minneapolis, MN USA) to stromal\ncells, the media was removed and cells were\nwashed with PBS and incubated with serum-free\nmedia overnight and then were treated with adiponectin\nat 0, 10, 100, and 200 ng/ml in 24, 48,\nand 72 hours for each dose ( 21 ).\nTo analyze the viability of cells, we used trypan\nblue staining. The stained and non-stained cells\nwere counted by hemocytometer and the percentage\nof the cells viability was calculated by dividing the number of non-stained cells by total number\nof cells multiplied by 100 ( 22 ).\nTotal RNA was extracted from stromal cells\nin control group and adiponectin group (100\nng/ml for 48 hours) using RNA purification kit\n(Jena Bioscience, GmbH, Germany). Total RNA\n(≥1μg) was used to synthesize complementary\nDNA (cDNA) in a 20 μl reaction by AccuPower\n® RocketScriptTM RT PreMix kit (BIONEER,\nKorea) and oligo(dT). The PCR was performed\nusing PCR PreMix kit (BIONEER, Korea) according\nto the manufacturer’s instructions.\nCycle conditions were as follows: initial denaturation\nat 94˚C for 10minutes; followed by 35\ncycles of denaturation at 94˚C for 60 seconds,\nannealing at 58˚C (GAPDH) and 62˚C) Adipo\nR1 and Adipo R2 (for 60 seconds and extension\nat 72˚C for 60 seconds, with a final extension at\n72˚C for 10 minutes ( Table 1 ). Since less than 35\ncycles produced PCR products at low intensity,\nthe PCR reactions were thought to be still in the\nexponential phase. Experiments were performed\nin triplicate to ensure reproducibility.\nThe expression of target genes was quantified\nagainst the internal reference gene (GAPDH). Products\nwere electrophoresed on a 1.5% agarose gel.\nGels were stained with ethidium bromide (10 μg/\nmL) and photographed on an ultraviolet transilluminator\n(UVIdoc; Uvitec, Cambridge, UK). Gel images\nwere analyzed using the UN-SCAN-IT program.\nSemi-quantitative RT-PCR values were presented\nas a ratio of the density of Adipo R1 and Adipo\nR2 bands divided by density of GAPDH bands. RTPCR\nwas performed as three individual replicates.\nData are reported as means ± SEM and statistical\nanalysis was done by SPSS (version 16) using one\nway analysis of variance (ANOVA) followed by\ntukey test. The significance of differences in expression\nof mRNA between two groups was determined\nusing the unpaired Student’s t test. P<0.05\nwas considered significant.\nCharacteristics of the primers used for target genes and internal control\n\nHuman endometrial stromal cells were treated\nwith adiponectin (10. 100, and 200 ng/ml) for 24,\n48, and 72 hours. Treatment with adiponectin decreased\nthe viability of stromal cells depending on\ndose and time ( Fig 1 ). 100 and 200 ng/ml doses in\nall of the experiment times indicated a significant\ndifference compared to control group, so cell viability\nin adiponectin (200 ng/ml) was 76.3% after\n72 hours (p<0.001) ( Fig 2 ). Adiponectin (10 ng/\nml) showed significant difference only in 48 and 72\nhours in comparison with control group (p<0.001).\nEqual numbers of cells were incubated for 48\nhours with various concentrations of 10 ng/ml (B), 100 ng/ml (C), and 200 ng/ml (D) adiponectin. As\nit is indicated, treatment with higher concentrations\nof adiponectin has resulted in a significant\ndecrease in cells viability in comparison with the\ncontrol group (A).\nEqual numbers of cells were exposed to adiponectin\n(10, 100, and 200 ng/ml) for 24, 48,\nand 72 hours and cells viability was examined\nby trypan blue staining method. Adiponectin decreased\ncell viability depending on dose and time\nand caused cell death. Columns marked with asterisk\nindicate the significant difference compared to\ncontrol group (p<0.001).\nMorphology of normal human endometrial stromal\ncells in the presence of adiponectin: Control group:\n(A ×100), 10 ng/ml group: (B ×100), 100 ng/ml group: (C\n×100), 200 ng/ml group: (D ×100).\nEffect of Adiponectin on viability of normal human\nendometrial stromal cell. Equal numbers of cells were exposed\nto adiponectin (10, 100, and 200 ng/ml) for 24, 48,\nand 72 hours and cells viability was examined by trypan\nblue staining method. Adiponectin decreased cell viability\ndepending on dose and time and caused cell death. Columns\nmarked with asterisk indicate the significant difference compared\nto control group (p<0.001).\nIn this study, expression of Adipo R1 and Adipo\nR2 in normal human endometrial stromal cells in\nthe secretory phase in the presence and absence of\nAdiponectin was demonstrated by semi-quantitative\nRT-PCR analysis ( Fig 3 ). The results revealed\nthat expression of Adipo R1 and Adipo R2 mRNA\nin control group (without Adiponectin) and treatment\ngroup (100 ng/ml adiponectin) for 48 hours\ndid not indicate significant difference (p>0.05).\nExpression of Adipo R1 and Adipo R2 in normal human\nendometrial stromal cells with and without Adiponectin\nin the secretory phase was demonstrated by semi-quantitative\nRT-PCR analysis. Expression of Adipo R1 and Adipo\nR2 mRNA in control group and treatment group (100 ng/ml\nadiponectin) did not indicate significant difference (p>0.05).\n\nIn our study, the  in vitro  effect of adiponectin\non viability of normal HESCs and expression of\nAdipo R1 and Adipo R2 receptors was examined.\nThe findings indicated that adiponectin depending\non dose and time decreased the viability of HESCs\nsignificantly. This finding, confirms the findings\nreported by Cong et al. which regard to the inhibitory\neffect of adiponectin on the endometrial carcinoma\ncell lines (HEC-1-A and RL95-2) in the\nculture ( 23 ) as well as anti-proliferative effects on\ntrophoblast cells and trophoblast cell lines (JEG-3 and BeWO) and decreasing their numbers in the\nculture ( 21 ).\nThe effects of adiponectin on cell death and\ndecreasing stromal cells count, in this study, was\nobserved with concentrations much lower than\nnormal level which is normally circulating in human\nblood serum ( 24 ,  25 ). Furthermore, the obtained\nresults in this study are compatible with\nthe findings of previous research regarding the\ndecreasing impact of adiponectin on the viability\nof various cancer cells such as breast cancer cell\nline (MCF7), prostate, endothelial cancer and bone\ncells ( 26 - 29 ).\nLower level of adiponectin is an independent\nrisk factor in the incidence of infertility and\nreproduction and different genital cancers in\nepidemiological studies. The direct and indirect\nmechanisms that influence this phenomenon are\nnot still well-known ( 30 ). However, it seems that\nadiponectin exerts its biological effects through\ntwo receptors named Adipo R1 and Adipo R2.\nTakemura et al. in 2006 showed the expression\nof two receptors of adiponectin in the epithelial\nand endometrial stromal cells of endometrial tissue\n( 31 ). The presence of these two receptors in\nvarious normal tissues and cancer cells has been\nconfirmed ( 32 - 34 ).\nIn the present study, the expression of Adipo R1\nand Adipo R2 mRNA in the absence of adiponectin\nas well as presence of 100 ng/ml adiponectin\nin normal stromal cells was analyzed. Expression\nof these two receptors in these cells was observed\nwhich confirms the findings of previous studies in\nwhich the presence of these receptors in endometrial\nstromal cells in both secretory and proliferative\nphases was demonstrated ( 31 ). However, the\nresults of our study revealed that adiponectin did\nnot have significant effect on the expression of\nAdipo R1 and Adipo R2 receptors.\nIn another study, the expression of Adipo R1 and\nAdipo R2 receptors in human normal endometrial\nand endometrial cancerous tissue in the presence\nof adiponectin ( in vitro ) was investigated. The\nfindings showed that adiponectin decreased cell\nproliferation in human endometrial cancerous tissue\nvia adiponectin receptors and the level of Adipo\nR1 expression was higher than that of Adipo\nR2 but the level of expression of receptors in cancerous\ntissue did not indicate significant difference\ncompared to normal non-cancerous tissue ( 30 ).\nThe recent research has indicated that expression\nof Adipo R1 in breast cancer cells ( 32 ) and human\nendometrial cancerous tissue ( 23 ) is higher than\nth at of Adipo R2.\nThese findings are compatible with the results\nof our study. The reason for this could be because\nof adiponectin binding to Adipo R1 and\nAdipo R2 receptors and the ability of these receptors\nin activating ligand-dependent AMP-activated\nprotein kinase (AMPK). Activation of AMPK results\nin decreasing cell proliferation and increasing\nthe number of inhibited cells in G 1 /G 0  phase and\nconsequently inducing cell death ( 35 ).\n\nAdiponectin inhibit endometrial stromal cell\nproliferation in dose and time dependant manner,\nand cause cell death. It can suggest as anti-endometriosis\nagent.\nFor further studies on the effect of adiponectin in\ninhibition of progressive development and proliferation\nof endometriotic cells, endometrial stromal\ncells of endometriosis patients should be used and\nthe function and expression of its receptors in the\ndevelopment of the disease must be investigated.","source_license":"public-domain-us","license_restricted":false}