Relationship between Insuline-like Growth Factor-I and Progesterone Secretion of Cultured Human Trophoblast Cells in Vitro

Objective To investigate the effect of insuline-like growth factor-Ⅰ （IGF-Ⅰ） on progesterone genesis and regulation. Methods Cytotrophoblast cells were collected by trypsin-collagenase digestion and percoll gradient centrifugation for primary culture. After stimulated with different concentrations（100 μg/ml, 10 μg/ml, 1 μg/ml, 0.1 μg/ml） of IGF-Ⅰ at the same time and with different duration（12 h,24 h,48 h, 72 h） of IGF-Ⅰ with the same concentration, progesterone levels in the media were measured by radioimmunoassay. Simultaneously, semiquantitative reverse transcriptase polymerase chain reaction （RT-PCR） was applied to determine the expression of low density lipoprotein receptor （LDLR） mRNA. Results Progesterone levels correlated positively with IGF-Ⅰ along with the IGF-Ⅰ concentration increasing, progesterone level began to increase at 12 h, and reached the climax at 48 h when cultured with 100 μg/L IGF-Ⅰ. The expression of LDLR mRNA was detectable in every group and accordant with variation of progesterone level. Conclusion Progesterone secretion has time- and dose-dependent effect on IGF-Ⅰ, and IGF-1 can up-regulate the expression of LDLR mRNA. IGF-Ⅰ may play an important role in promoting secretion of progesterone in trophoblast cells.

Overexpression of insulin-like growth factor-Ⅰ receptor as a pertinent biomarker for hepatocytes malignant transformation

AIM:To investigate the dynamic features of insulinlike growth factor-Ⅰreceptor(IGF-ⅠR)expression in rat hepatocarcinogenesis,and the relationship between IGF-ⅠR and hepatocytes malignant transformation at mRNA or protein level.METHODS:Hepatoma models were made by inducing with 2-fluorenylacetamide(2-FAA)on male SpragueDawley rats.Morphological changes of hepatocytes were observed by pathological Hematoxylin and eosin staining,the dynamic expressions of liver and serum IGF-ⅠR were quantitatively analyzed by an enzymelinked immunosorbent assay.The distribution of hepatic IGF-ⅠR was located by immunohistochemistry.The fragments of IGF-ⅠR gene were amplified by reverse transcription-polymerase chain reaction,and confirmed by sequencing.RESULTS:Rat hepatocytes after induced by 2-FAA were changed dynamically from granule-like degeneration,precancerous to hepatoma formation with the progressing increasing of hepatic mRNA or IGF-ⅠR expression.The incidences of liver IGF-ⅠR,IGF-ⅠR mRNA,specific IGF-ⅠR concentration(ng/mg wet liver),and serum IGF-ⅠR level(ng/mL)were 0.0%,0.0%,0.63±0.17,and 1.33±0.47 in the control;50.0%,61.1%,0.65±0.2,and 1.51±0.46 in the degeneration;88.9%,100%,0.66±0.14,and 1.92±0.29 in the precancerosis;and 100%,100%,0.96±0.09,and2.43±0.57 in the cancerous group,respectively.IGF-ⅠR expression in the cancerous group was significantly higher(P<0.01)than that in any of other groups at mRNA or protein level.The closely positive IGF-ⅠR relationship was found between livers and sera(r=0.91,t=14.222,P<0.01),respectively.CONCLUSION:IGF-ⅠR expression may participate in rat hepatocarcinogenesis and its abnormality should be an early marker for hepatocytes malignant transformation.

The effect of polymorphism in gene of insulin-like growth factor-Ⅰ on the serum periparturient concentration in Holstein dairy cows

Objective:To investigate the relationship between polymorphism within the 5'-untranslated region(5'-UTR)of IGF-I gene and its periparturient concentration in Iranian Holstein dairy cows.Methods:Blood samples(5 mL,n=37)were collected by caudal venipuncture from each animal into sample lubes containing the EDTA and DNA was extracted from blood.In order to measure ICF-I concentration the collection of blood samples(n=111)was also done at 14 d before calving(prepartum),25 and 45 d postpartum.Results:We found evidence for a significant effect of C to T mutation in position 512 of IGF-I gene on its serum concentration in dairy cows in Iran.Cows with CC genotype had significantly higher concentration(Mean-SD)of IGF-I at 14 d prepartum(91.8±18.1)μg/L compared to those with TT genotype(73.3±14.4)μg/L(P=0.04).A significant trend(quadratic)was found for IGF-I concentration,as higher in CC cows compared to ones with TT genotype,during the 14 d before calving to 45 d postpartum(P=0.01).Conclusions:We concluded that C/T transition in the promoter region of IGF-I gene can influence the serum concentration of ICF-I in periparturient dairy cows.

Expression of Connective Tissue Growth Factor in Renal Tubulointerstitial Fibrosis in Rats and Its Pathogenic Role

Summary： In order to explore the role of connective tissue growth factor （CTGF） in the pathogenesis of renal tubulointerstitial fibrosis, 48 Wistar rats were randomly divided into sham-operated and unilateral ureteral obstruction （UUO） group. On the postoperative day 1, 3, 7 and 14, the rats were killed and the kidneys were removed. The renal tubulointerstitial injury index was evaluated according to the MASSON staining. The mRNA levels of CTGF, transforming growth factor β1 （TGF-β1）. collagen [ （col I ）, and plasminogen activator inhibitor-1 （PAI 1） were detected using rexerse transcriptional-polymerase chain reaction （RT PCR）. Immunohistochemistry was performed to evaluale the protein expression of the above factors, and the relations among them were analyzed. Quantitative expression of CTGF protein in the kidneys was also assessed using Western blot. The results showed that TGF-β1 mRNA level was increased at first day after UUO, followed by a marked elevation of CTGF mRNA level, which began to increase 3 days after UUO （P〈0.01）. With the progression of the disease, the mRNA expression of CTGF, col I and PAI-1 was increased progressively. Immunohistochemistry revealed that the CTGF protein expression was significantly increased in fibrotic areas and tubular epithelial cells 3 days after UUO. On the post-UUO day 7, the protein level of CTGF was positively related to the renal tubulointerstitial injury index （r =0.62, P〈0.01）, the expression of TGF-β1 （r=0.85, P〈0.01）, colI （r=0.78, P〈0.01）, and PAI-1（r=0.76, P〈0.01）. Upon Western blot analysis, CTGF protein expression began to increase 3 days after UUO, and appeared progressively throughout the time course （P〈0.01, as compared with sham-operated group）. It is concluded that CTGF can be induced by TGF-β and mediate various profibrotic actions of this cytokine, such as increasing extracellular matrix （ECM） synthesis and decreasing ECM degradation. The increased expression of CTGF may play a crucial role in the development and progression of tubulointerstitial fibrosis.

The expression of insulin-like growth factor-Ⅰ mRNA and polypeptide in rat osteoblasts with exposure to parathyroid hormone

Objective To investigate the insulin-like growth factor-Ⅰ (IGF-Ⅰ) gene and polypeptide expression in cultured rat osteoblast (ROB) and the role of IGF-Ⅰ in mediating the cell-to-cell communication by mimicking the pharmacokinetics of parathyroid hormone (PTH). Methods The ROB was cultured with three kinds of treatment: (1) Control (Ctr), the cells were cultured without PTH during the first 6 hours and the subsequent 42 hours in a 48-hour cycle; (2) Intermittent exposure to PTH (Itm), the cells were cultured with PTH during the first 6 hours, but without PTH in the subsequent 42 hours; and (3) Continuous exposure to PTH (Ctu), the cells were cultured with PTH during the first 6 hours and the subsequent 42 hours. Results The bone-forming activities of ROB were increased in Itm and inhibited in Ctu. The IGF-Ⅰ mRNA content in Itm cells was elevated only during the first 6 hours and that in Ctu cells was elevated at any time during an incubation cycle. The free IGF-Ⅰ concentration in the medium of Itm cells was generally higher and that of the Ctu cells was generally lower compared with those of the Ctr cells. The IGF-Ⅰ antibody significantly reduced the alkaline phosphatase activity within the cells of Ctr and Itm. Conclusions PTH rapidly and constantly stimulates the IGF-Ⅰ gene transcription of osteoblast. There was an obvious discrepancy between the IGF-Ⅰ mRNA content within the osteoblast and the free IGF-Ⅰ level around the osteoblast in either mode of PTH action. The IGF-Ⅰ might be important for osteoblast-osteoblast communication and bone-forming activity, not only in intermittent PTH administration, but also in the physiological functioning of osteoblasts.

Gardenia jasminoides attenuates hepatocellular injury and fibrosis in bile duct-ligated rats and human hepatic stellate cells

AIM:To investigate the anti-hepatofibrotic effects of Gardenia jasminoides in liver fibrosis.METHODS:Male Sprague-Dawley rats underwent common bile duct ligation(BDL) for 14 d and were treated with Gardenia jasminoides by gavage.The ef-fects of Gardenia jasminoides on liver fibrosis and the detailed molecular mechanisms were also assessed in human hepatic stellate cells(LX-2) in vitro.RESULTS:Treatment with Gardenia jasminoides decreased serum alanine aminotransferase(BDL vs BDL + 100 mg/kg Gardenia jasminoides,146.6 ± 15 U/L vs 77 ± 6.5 U/L,P = 0.0007) and aspartate aminotransferase(BDL vs BDL + 100 mg/kg Gardenia jasminoides,188 ± 35.2 U/L vs 128 ± 19 U/L,P = 0.005) as well as hydroxyproline(BDL vs BDL + 100 mg/kg Gardenia jasminoides,438 ± 40.2 μg/g vs 228 ± 10.3 μg/g liver tissue,P = 0.004) after BDL.Furthermore,Gardenia jasminoides significantly reduced liver mRNA and/or protein expression of transforming growth factor β1(TGF-β1),collagen type?Ⅰ?(Col?Ⅰ) and α-smooth muscle actin(α-SMA).Gardenia jasminoides significantly suppressed the upregulation of TGF-β1,Col?Ⅰand α-SMA in LX-2 exposed to recombinant TGF-β1.Moreover,Gardenia jasminoides inhibited TGF-β1-induced Smad2 phosphorylation in LX-2 cells.CONCLUSION:Gardenia jasminoides exerts antifibrotic effects in the liver fibrosis and may represent a novel antifibrotic agent.

Significance of changes in transforming growth factor-β mRNA levels in autogenous vein grafts

Background This study was designed to investigate changes in mRNA levels of transforming growth factor-β(TGF-β), collagen Ⅰ, and collagen Ⅲ in autogenous vein grafts. Methods Twenty-four New Zealand rabbits were randomly divided into 4 groups with 6 rabbits each. The external jugular veins of the New Zealand rabbits were harvested and grafted into the ipsilateral carotid artery. All rabbits were fed with a standard diet. After the operation, the rabbits were sacrificed at 1, 2, 3, or 4 weeks. TGF-β, collagen Ⅰ, and collagen Ⅲ mRNA levels in the venous grafts were measured by semiquantitative methods at every time point. The contralateral external jugular veins were also harvested and analyzed as controls. Glyceraldehyde-3-phosphate dehydrogenase was used as an internal standard to normalize all samples for potential variations in mRNA content. In order to observe the expression of TGF-β protein, immunohistochemical SABC methods were used. Results One week postoperation, the mRNA level of TGF-β was upregulated to 1.73±0.19 in the vein graft and 1.21±0.16 in the control vein (P<0.01). High mRNA levels were maintained until week 4 postoperation. The mRNA levels of collagen Ⅰ and collagen Ⅲ were also significantly increased to 2.18±0.21 versus 1.12±0.24 and 1.08±0.13 versus 0.83±0.12, respectively (P<0.05). Immunohistochemical staining revealed a higher density of TGF-β expression in the vein grafts.Conclusions An uninterrupted increase in mRNA levels of TGF-β, collagen Ⅰ, and collagen Ⅲ is observed in autogenous vein grafts. This increase may be the major cause of intimal hyperplasia, sclerosis, and even graft failure.

Branched-chain amino acids partially recover the reduced growth of pigs fed with protein-restricted diets through both central and peripheral factors

The objective of this study was to assess the growth efficiency of pigs fed with protein-restricted diets supplemented with branched-chain amino acids(BCAA)and limiting amino acids(LAA)above the rec-ommended levels.Following 2 weeks of adaptation,48 young barrows were weight matched and randomly assigned to 6 treatments(8 pigs/treatment)for 4 weeks:positive control(PC)with standard protein,negative control(NC)with very low protein containing LAA(i.e.,Lys,Met,Thr and Trp)at rec-ommended levels,and NC containing LAA 25%(L25),LAA 50%(L50),LAA+BCAA(i.e.,Leu,Ile and Val)25%(LB25)and LAA+BCAA 50%(LB50)more than recommendations.Feed intake(FI)and body weight(BW)were measured daily and weekly,respectively.At week 6,blood samples were collected,all pigs euthanized and tissue samples collected.The data were analyzed by univariate GLM or mixed procedure(SPSS)and the means were separated using paired Student's t-test followed by Benjamini-Hochberg correction.Relative to PC,NC had decreased FI,BW,unsupplemented plasma essential amino acids,serum insulin-like growth factor-I(IGF-I)and hypothalamic neuropeptide Y(NPY)(P<0.01).Compared to NC,L25 or L50,LB50 had increased BW and serum IGF-I and decreased plasma serotonin and both LB25 and LB50 had higher FI,plasma BCAA,hypothalamic 5-hydroxytryptamine-receptor 2A and NPY and jejunal 5-hydroxytryptamine-receptor 7(P<0.01).Overall,supplementation of protein-restricted diets with increased levels of dietary BCAA partially recovered the negative effects of these diets on growth through improved IGF-I concentration and FI,which was associated with changed expression of serotonin receptors,blood AA and hypothalamic NPY.