Expression of Insulin-like Growth Factor-I in Uterus is Ovarian Steroid Dependent： An in situ Immunohistochemical Study in Rat

Objective To study the immunohistochemical localization of insulin-like growth factor-I (IGF-I) in rats’uteri to determine if expression of the growth factor is ovarian steroid dependent. Method The study was carried out in presence and absence of ovary in situ of non pregnant females and during early gestation (day 1 to day 5.5). Cyclic females were tested to observe the effect of native steroids on IGF-I expression. Adult females were ovariectomized (OVX) and injected (s.c) with estradiol-17β in a dose of 0.1μg/ml per day for three consecutive days at interval of 24h prior to the collection of uterine horns. During the early pregnancy, studies were carried out on day 3.5 and day 5.5 of geatation respectively to determine the steroids’effects during pre- and post-implantation period. Tamoxifen was administered (s.c) in a dose of 250μg/ml per day from day 1 to day 3 of gestation while, the prostaglandin F 2α (PGF 2α ) was administered (s.c) from day 3 of gestation onward for three consecutive days at interval of 24h in a dose of 150μg/ml per day. Expression of IGF-I was immunohistochemically localised using IGF-I antibody in paraffin embedded sections. Results IGF-I was expressed in rat uterus during estrus phase as well as during pre and post-implantation period. The ovariectomized females’uteri lost the expression of IGF-I. Exogenous administration of tamoxifen and PGF 2α reduce the expression of the growth factor. Conclusion Expression of IGF-I in rat uterus during cyclic stage and early gestation depends upon the availability of circulating estrogen and progesterone. Uterine expression of IGF-I can be modulated by manipulating circulating ovarian steroid either during cyclic stage or during gestation.     

Root Extract of Polygonum Hydropiper Alters the Expression of Rat Uterine Protein Profile in Presence and Absence of Ovary in-situ during Periimplantation Period: An Evidence on SDS-PAGE

Objective To study its effects on reproductive performance in albino rats.
Methods The chromatographic fraction （CF） of crude methanolic extract of the herb has been subcutaneously administered to female albino rats. Experiments were carried out in adult cyclic females and oavriectomised （OVX） females during early gestation period. Uterine horns were collected following the respective treatment regimen to stud）, the protein profile in 15% gel SDS-PAGE.
Results The CF induced changes in the expression of protein in rat uterus. New proteins have been expressed in uterus of adult cyclic females having ovary in-situ. The OVX females treated with CF showed altered uterine protein profile compared with that of OVX control and OVX estradiol-17β （E2） treated rats. The CF exerted its effect on expression of uterine protein during early gestation period in rats. While uterine proteins of CF treated females were similar to that of controls during preimplantation period; many of the proteins on day 6 of gestation have been found either missing or expressed in lesser intensity.
Conclusion The root of Polygonum hydropiper contains potential compound（s） which can alter the reproductive performance of female rats modulating uterine protein expression.     

Effect of ethanol extract of Rhodiola rosea on the early nephropathy in type 2 diabetic rats

This study aimed to investigate the therapeutical effects of Rhodiola rosea extract on rats with type 2 diabetic nephropathy (DN). The rat type 2 DN model was established by high fat and high calorie feeding and intravenous injection of streptozocin (STZ). Wistar rats were randomly divided into normal group, control group, low dose Rhodiola rosea group, high dose Rhodiola rosea group and Captopril group. Oral glucose tolerance test (OGTT) was performed to determine the impairment of glucose tolerance in the established animal model. A series of parameters including fasting blood glucose (FBG), total cholesterol (TC), triglyceride (TG), creatinine clearance rate (Ccr), 24-h urinary albumin (UA), the ratio of kidney mass/body weight (renal index) and glomerular area were examined after 8 weeks. Moreover, the expression of transforming growth factor (TGF)-β1 in renal tissues was detected by using immunohistochemisty. At the end of the eighth week, FBG, TC, TG, Ccr, 24-h urinary albumin, the ratio of kidney mass/body weight and glomerular area were significantly reduced in Rhodiola rosea extract treatment groups as compared with those in control group. TGF-β1 expression in renal tissues of Rhodiola rosea extract treatment groups was also significantly decreased as compared with that of control group. These results indicate that Rhodiola rosea extract may have a protective effect on early nephropathy in diabetic rats, which might be related to the decrease of the renal expression of TGF-β1.     

Effects of Houttuynia Cordata Thumb on Expression of BMP-7 and TGF-β1 in the Renal Tissues of Diabetic Rats

Objective： To explore the effects of Houttuynia Cordata Thumb （HCT 鱼腥草 Yu Xing Cao） on expression of transforming growth factor-β1 （TGF-β1） and bone morphogenetic protein-7 （BMP-7） in the renal tissues of diabetic rats. Methods： The diabetic rats induced by intravenous injection of streptozotocin（STZ） were randomly divided into a model group, a HCT group and a lotensin group, with normal rats designated as the controls. 8 weeks later, the ratio of kidney weight to body weight, the glomerular area, the excretion of β 2-microglobin （β2-MG） in 24-hr urine, the albumin excretion in 24-hr urine, and creatinine clearance rate （CCR） were investigated. The expression of TGF- β 1, BMP-7 and collagen I in the renal tissues was observed with the immunohistochemical method and by the semi-quantitative assay. Results： The overgrowth of glomerulus, the excretion of β 2-MG in 24-hr urine, the albumin excretion rate in 24-hr urine and CCR in the HCT group significantly reduced （P〈0.05）, and the expression of TGF-β1 and collagen I significantly decreased （P〈0.05）, but BMP-7 significantly increased （P〈0.05） in the HCT group as compared with those in the model group, with no significant difference as compared with the lotensin group （P〉0.05）. Conclusion： HCT has a protective effect on the renal tissues in diabetic rats, which is probably correlated with the decrease of the expression of TGF-β1 and collagen I and with the increase of the expression of BMP-7 in the renal tissues.     

Caffeine Exposure Causes Immune Dysfunction and Intrauterine Growth Restriction Retardation in Rats

<正>This study was conducted to assess the effects of caffeine on the dam and the physical development of rat offspring.Pregnant Sprague-Dawley rats (20per dose group) were administered caffeine by gavage at 0 (control),5,20,and 80 mg/kg body weight bw daily from gestational day 6 through lactation using caffeine dissolved in water.The developmental toxicity of caffeine was evaluated.     

Effect of Hydro-ethanolic （1：1） Extract of Stephania Hernandif olia and Achyranthes Aspera in Composite Manner on Testicular Activity in Male Albino Rat： ADose Dependent Analysis

Objective To investigate the effect of hydro-ethanolic extract of Stephania hernandifolia leaves and Achyranthes aspera roots in composite manner at the ratio of 1:3 on testicular activity in male rats. Methods Rats were divided into 4 groups with 8 animals in each group. The control (group A) received 0.5ml of olive oil/100g body weight orally, other three groups were treated with said extract orally at a dose of 0.4mg/g (group B) or 0.8mg/g (group C) or 1.6mg/g body weight (group D) respectively for 28d. On 29th day of experiment, the animals were sacrificed. Sperm concentrations in cauda epididymis and biochemical markers like testicular cholesterol, androgenic key enzyme activity, plasma testosterone level, seminal fructose level, glutamate oxaloacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT) activities and serum triglyceride levels were measured following standard methods. Results Extract treated animals in all doses resulted in a significant (P<0.05) decrease in sperm concentration, testicular androgenic key enzyme activity, plasma testosterone and seminal vesicle fructose levels along with an increase in testicular cholesterol level. Animals treated at a dose of 0.8mg/g body weight showed more promising result without causing any metabolic toxicity compared with other doses. Histological study also supported the biochemical results. The minimum but most effective dose i.e. 0.8mg/g body weight had an inhibitory effect on implantation focused here by mating experiment.Conclusion The composite extract of S.hernandifolia and A. aspera has a potent efficacy as male contraceptive that may provide clues to the pharmaceutical industries for male contraceptive development.     

Verification on the Developmental Toxicity of Short-term Exposure to Phenol in Rats

Objective To verify the health advisory for short-term exposure to phenol.Methods The method of this validation experiment was the same as the US Environmental Protection Agency(EPA) methodology for toxicology experiments used to determine phenol drinking water equivalent level(DWEL). Pregnant female Sprague-Dawley rats were administered phenol in distilled water by gavage at daily doses of 15, 30, 60, 120, and 240 mg/kg body weight(b.w.)from implantation(the 6 th day post-mating) to the day prior to the scheduled caesarean section(the20 th day of pregnancy). The following information was recorded: general behavior; body weight;number of corpus luteum, live birth, fetus, stillbirth, and implantation; fetal gender; body weight; body length; tail length; and abnormalities and pathomorphological changes in the dams.Results In the 60 mg/kg b.w. dose group, the mortality of pregnant rats increased with increasing doses, suggesting maternal toxicity. Fetal and placental weights decreased as phenol dose increased from30 mg/kg b.w., and were significantly different compared those in the vehicle control group, which suggested developmental toxicity in the fetuses. However, the phenol-exposed groups showed no significant change in other parameters compared with the vehicle control group(P 0.05).Conclusion Despite using the same method as the US EPA, a different NOEAL of 15 mg/(kg·d) was obtained in this study.     

Role of BMP-7 and TGF-β1 expression in renal tubulo-interstitial lesion of Wistar rats induced by unilateral ureteral obstruction

Objective： To explore the role of bone morphorgenetic protein-7 （BMP-7） in the renal tubulo-interstitial lesions induced by unilateral ureteral obstruction （UUO）. Methods： Sixty Wistar rats were equally and randomly divided into normal control, sham operation and UUO groups, and respectively sacrificed on the 1st, 3rd, 7th, 14th day after the time of UUO operation. The mRNA levels of BMP-7 and TGF-β1 in the renal tissues were examined by RT-PCR. The expression sites and levels of BMP-7 and TGF-β1 proteins were detected by immunohistochemistry staining. Results：Compared to control groups, the level of BMP-7 mRNA was significantly decreased, but that of TGF-β1 mRNA was significantly increased in UUO rats. Immunohistochemistry staining indicated that BMP-7 mainly expressed in the renal tubules and interstitum, rarely in the glomeruli. In UUO rats, the expression of BMP-7 protein was decreased, but that of TGF-β1 was increased in an obstruction dependent manner. Conclusion：The downregulation of BMP-7 is observed in the early phase of fibrotic process of the renal interstitium, suggesting it may be involved in the formation and development of the tubulo-interstitial lesions.     

Role of reactive oxygen species in triptolide-induced apoptosis of renal tubular cells and renal injury in rats

This study investigated the role of reactive oxygen species(ROS) in the pathogenesis of triptolide-induced renal injury in vivo.Rats were randomly divided into 4 groups(n=5 in each):triptolide group in which the rats were intraperitoneally injected with triptolide solution at a dose of 1 mg/kg of body weight on day 8;control group in which the rats received a single intraperitoneal injection of 0.9% physiological saline on day 8;vitamin C group in which the rats were pretreated with vitamin C by gavage at a dose of 250 mg/kg of body weight per day for 7 days before the same treatment as the control group on day 8;triptolide+vitamin C group in which the rats were first subjected to an oral administration of vitamin C at a dose of 250 mg/kg of body weight per day for 7 days,and then to the same treatment as the triptolide group on day 8.All the rats were sacrificed on day 10.Blood samples were collected for detection of plasma creatinine(Pcr) and plasma urea nitrogen(PUN) concentrations.Both kidneys were removed.The histological changes were measured by haematoxylin-eosin(HE) staining.The production of ROS was determined by detecting the fluorescent intensity of the oxida-tion-sensitive probe rhodamine 123 in renal tissue.Renal malondialdehyde(MDA) content was meas-ured to evaluate lipid peroxidation level in renal tissue.TUNEL staining was performed to assess apop-tosis of renal tubular cells.Renal expression of apoptosis-related proteins Bcl-2,Bax,Bid,Bad,Fas and FasL,as well as corresponding encoding genes were assessed by Western Blotting and real-time PCR.The results showed that triptolide treatment promoted the generation of a great amount of ROS,up-regulated the expression of Bax,Bid,Bad,Fas and FasL at both protein and mRNA levels,as well as the ratio of Bax to Bcl-2,and caused the apoptosis of renal tubular cells and renal injury.However,pretreatment with an antioxidant,vitamin C,significantly reduced the generation of ROS and effectively inhibited the triptolide-induced apoptosis of renal tubular cells and renal injury.It was concluded that ROS plays a critical role in triptolide-induced apoptosis of renal tubular cells and renal injury.The protective administration of vitamin C may help alleviate triptolide-induced renal injury and nephrotoxicity.     

Expression of Ceramide Kinase （Cerk） and Related Regulation in Early Pregnant Uterus of Mice

Objective To investigate the expression of ceramide kinase (Cerk) and related regulation in mouse uterus during early pregnancy. Methods Several mouse models, including early pregnancy, pseudopregnancy, delayed or activated implantation, artificial decidualization, and steroid hormonal treatment were performed (n=10). Immunohistochemistry and in situ hybridization were used to detect the expression of Cerk protein and mRNA in uterus. Results Expression of Cerk mRNA and protein was strongly detected in the luminal and glandular epithelium on day 1 of pregnancy. However, Cerk mRNA and protein signals were strongly detected in the subluminal stroma surrounding the implanting blastocyst on day 5 and decidua from day 6 to day 8 whereas not in the luminal epithelium. The expression of Cerk in luminal and glandular epithelium of pseudopregnancy was similar to that of early pregnancy from day 1 to day 4 whereas on day 5 of pseudoprgenancy still with remarkable signals in the luminal epithelium. Under delayed implantation, no obvious Cerk expression was detected in the uterus. After delayed implantation was terminated by estrogen treatment and embryo implantation was initiated, both Cerk mRNA and protein were detected in the subluminal stroma surrounding the implanted blastocyst. A strong Cerk signal was detected in decidualized cells under artificial decidualization, whereas only a basal level of Cerk signal was observed in the control uterus which did not inject sesame oil. Progesterone induced a slight expression of Cerk in the luminal and glandular epithelium. Both estradiol and a combination of progesterone with estradiol strongly increased the level of Cerk signal in the luminal and glandular epithelium. Conclusion Cerk expression is under the regulation of progesterone and estrogen. The strong expression of Cerk in implantation site and decidua suggests that Cerk might play an important role during implantation and decidualization.     

Effect of Bushenantai Recipe on the Expression of Endometrial LIF in Mice with Embryonic Implantation Dysfunction

In order to observe the effect of Bushenantai recipe on the expression of endometrial leukemia-inhibitory factor （LIF） in mice with embryonic implantation dysfunction （EID）, 120 Kunming mice post coition were randomized into three groups： normal control group, model group and traditional Chinese medicine group （TCM group） （n=40 in each group）. Uterus was collected on the pregnancy day （Pd） 4, 5, 6 after an intravenous injection of Evan＇s blue. The endometrium was dyed by Evan＇s blue and the mean points of response were observed on Pd 5. The expression of LIF mRNA and protein was detected by RT-PCR and immunohistochemistry respectively and analyzed statistically by image system. The results showed that the number of implantation sites in model group was remarkably less than in normal control group and TCM group. There was no significant difference between normal control group and TCM group. The expression of LIF mRNA and protein in model group was delayed. Bushenantai recipe could increase the expression of LIF mRNA and protein in endometria of mice with EID. It was suggested that Bushenantai recipe could improve embryo implantation of mice with EID by promoting the endometrial LIF expression and endometrial decidualization.     

Expression of Angiopoietin-1/-2 in the Process of Mouse Embryo Implantation

This study examined the expression and distribution of angiopoietin-1/-2 (Ang-1/-2) in the endometrium of early pregnant mice. The expression of Ang-1/-2 was detected by immunohisto- chemical staining and in situ hybridization respectively. Computerized image analysis system was used to measure the average optical intensity of Ang-1/-2 in endometria at different time points after gestation. Mice were randomly divided into 5 groups: control group, D2 group (2 days after preg- nancy), D4 group (4 days after pregnancy), D6 group (6 days after pregnancy) and D8 group (8 days after pregnancy), each containing 15 mice. The results showed that the expression of Ang-1 and Ang-2 was very different among 4 groups (P<0.01). Immunohistochemical staining revealed that Ang-1 was localized in the cytoplasma of stromal cells 2 days after pregnancy (day 2), and in luminal epithelial cells on day 4. The protein of Ang-2 was mainly expressed in the cytoplasma of glandular epithelia and stromal cells. With gestation time, the positive reactions of Ang-1/-2 were stronger in the endometria of the pregnant mice (P<0.01). In situ hybridization showed Ang-1 mRNA in stromal cells on day 2. Hybridization signal was localized in both stromal cells and vessel epithelial cells on day 4; Ang-2 mRNA was expressed in stromal cells and glandular epithelia on day 2; high mRNA levels appeared in stromal cells, glandular epithelia and vascular endothelia on day 4; an increasing in mRNA expression of Ang-1/-2 was observed on day 6 and day 8 (P<0.01). It is suggested that Ang-1/-2 may play an important role in the cross-talk between blastocyst and maternal endometrium during the process of embryo implantation.     

Effect of Rapamycin on TGF-β1- induced epithelial-mesenchymal transition in LoVo colonic adenocarcinoma cells

Objective:To investigate the effect of Rapamycin on epithelial-mesenchyrnal transition(EMT) of LoVo colonic adenocarcinoma cells in vitro.Methods:Cultured LoVo colonic adenocarcinoma cells were divided into three groups: negative control group,EMT-inducing group(TGF-β1) and EMT-interfering group(TGF-β1 plus Rapamycin).E-cadherin expression in LoVo cells was detected by Western Blot,while the expression of vimentin was evaluated through immunocytochemistry.The Snail mRNA in LoVo cells was examined by RT-PCR.Results:TGF-β1 induced LoVo cell switching from polygonal to spindle-shaped.TGF-β1 enhanced the expression of vimentin,but lowered the level of E-cadherin.In contrast,Rapamycin impaired the transition induced by TGF-β1.Rapamycin dramatically abrogated TGF-β1-induced vimentin expression and restored E-cadherin expression in LoVo cells.Rapamycin significantly repressed the up-regulation of Snail mRNA expression induced by TGF-β1.Conclusion:Rapamycin dramatically abrogated TGF-β1 induced Snail mRNA expression in LoVo cells,hence inhibiting EMT of these cells in vitro.     

The Effects of Signal Protein SMADs on Rat Cardiocyte Hypertrophy

To investigate the role of signal protein hypertrophy were produced by constriction of the SMADs in rat cardiac hypertrophy. The rat models of cardiac abdominal aorta. The left ventricular mass index （LVMI） was investigated. The expression of transforming growth factor-β1 mRNA （TGF-β1） and Smad2, 3, 7mRNA were assessed by RT-PCR. The LVMI and the expression of TGF-β1 and Smad2, 3, 7mRNA in hypertrophic left ventricle were increased on the 3rd day after the operation and continued to the 4th week. The peak expression of TGF-β1 and Smad2, 3mRNA were in the 2nd week after operation. The expression of Smad7 began to increase in the 3rd day after operation, but the peak was in the 1st week after operation, and then decreased. The FGF-β1 and signal protein Smad2, 3, 7 were included in the progress of rat cardiac hypertrophy produced by constriction of abdominal aorta.     

Expression of Serum and Glucocorticoid-inducible Kinase1 in Diabetic Rats and Its Modulation by Fluvastatin

The expression of serum and glucocorticoid-induced protein kinase in the renal cortex of diabetic rats was examined, and the function of signal transduction mediated by SGK1 in diabetic nephropathy and its modulation by fluvastatin were also investigated. 24 male Wistar rats were randomly divided into normal control group （n = 8）, diabetic nephropathy group （n = 8） and fluvastatin-treated diabetic nephropathy group （15 mg/kg/d, n=8）. The metabolic parameters were measured at the 8th week. The expression of transforming growth factor β1 （TGF-β1） and fibronectin （FN） was immunohistochemically examined. The expression of SGK1 was detected by RT-PCR and Western blot, and CTGF mRNA was assessed by RT-PCR. As compared to DN, blood glucose, 24-h urinary protein, Cer and kidney weight index were all decreased and the weight was increased obviously in group F. At the same time, mesangial cells and extracellular matrix proliferation were relieved significantly. The levels of cortex SGK1 mRNA and protein were up-regulated, and both TGF-β1 and FN were down-regulated by fluvastatin. The mRNA of SGK1 was positively correlated with the CTGF, TGF-β1 and FN. SGK1 expression is markedly up-regulated in the renal cortex of DN group and plays an important role in the development and progress of diabetic nephropathy by means of signal transduction. Fluvastatin suppressed the increased SGKlmRNA expression in renal cortex and postponed the development of diabetic nephropathy.     

COA: Bone Morphogenetic Protein 2 Promoted Transforming Growth Factor β3 Induced Chondrogenesis of Human Osteoarthritic Synovium-Derived Stem Cells

Background Synovium-derived stem cells (SDSCs) with greater chondrogenic potential are attracting more considerable attention as a cell source for cartilage regeneration. The aim of this study was to investigate the effect of bone morphogenetic protein-2 (BMP-2) on transforming growth factor-beta3 (TGF-β3)-induced chondrogenesis of SDSCs isolated from human osteoarthritic synovium in a pellet culture system. Methods Nucleated cells isolated from human osteoarthritic synovium were plated at an optimal cell density to allow the selective proliferation of SDSCs. The clonogenicity, stem cell marker expression and multi-differentiation potential were determined by CFU assay, flow cytometry assay and specific staining including alizarin red S staining, Oil red staining and alcian blue staining, respectively. SDSCs pellet was cultured in chondrogenic medium without or with TGF-β3 or/and BMP-2. At day 21, the diameter and the weight of the pellets were measured. Chondrogenic differentiation of SDSCs was evaluated by Safranin O staining, immunohistochemical staining of collagen type II, sulfated glycosaminoglycan (sGAG) synthesis and mRNA expression of collagen type II (COL2A1), aggrecan (ACAN), SOX9, link-protein (HAPLN1), collagen type X (COL10A1) and BMP receptor II (BMPR-II). Results Cells isolated under the optimized culturing density (104/60cm2) showed clonogenicity and multi-differentiation potential. These cells were positive (>99% positive) for CD44, CD90, CD105 and negative (<10% positive) for CD34 and CD71. SDSCs differentiated to a chondrocytic phenotype in chondrogenic medium containing TGF-β3 with or without BMP-2. Metachromatic staining of the extracellular matrix with Safarnin O was positive and the expression of collagen type II was detected. The combination of TGF-β3 and BMP-2 produced cell pellets with larger diameter and weight, produced more sGAGs, expression higher levels of collagen type II and chondrogenic markers, except COL10A1, than medium with TGF-β3 alone. Conclusions SDSCs could be isolated from human osteoarthritic synovium. Supplementation of BMP-2 significantly promoted the in vitro TGF-β3-induced chondrogenic differentiation of SDSCs.