scholarly journals hUMSCs regulate the differentiation of ovarian stromal cells via  TGF-β1 /Smad3  signaling pathway to inhibit ovarian fibrosis to repair ovarian function in POI rats

2020 ◽  
Author(s):  
Linlu Cui ◽  
Hongchu Bao ◽  
Zhongfeng Liu ◽  
Xuejing Man ◽  
Hongyuan Liu ◽  
...  
Keyword(s):  
Western Blot ◽  
Signaling Pathway ◽  
Stromal Cells ◽  
Collagen Type ◽  
Ovarian Function ◽  
Ovarian Tissue ◽  
Ovarian Insufficiency ◽  
Tissue Fibrosis ◽  
Function Recovery ◽  
Tgf Β1

Abstract Objective: The basic pathological changes of primary ovarian insufficiency (POI) include ovarian tissue fibrosis and follicular development disorders. The human umbilical cord mesenchymal stem cells (hUMSCs) transplantation has been shown an effective method to improve the ovarian function in POI rat model; however, the exact mechanisms are still unclear. The purpose of this study is to investigate whether the recovery of ovarian function in POI rats is related to the inhibition of tissue fibrosis following hUMSCs transplantation. Furthermore, the transforming growth factor-β1 (TGF-β1 ) signaling pathway is explored to determine the mechanisms of ovarian function recovery through its inhibition of tissue fibrosis. Methods: The primary ovarian insufficiency (POI) rat model was established by intraperitoneal injection of chemotherapy drug cisplatin (CDDP) for 7 days, The levels of serum sex hormones were measured using enzyme-linked immunosorbent assay (ELISA). The tissue fibrosis in ovary was examined using masson staining and Sirus red staining. The collagen fibers in the ovarian tissues were detected by Western blot analysis. To investigate the mechanisms of ovarian function recovery following hUMSCs transplantation, ovarian stromal cells were isolated from the ovarian cortex of immature rats. The expression of Cytochrome P450 17A1 (Cyp17a1) and fibrosis marker of alpha smooth muscle Actin (α-SMA) in ovarian stromal cells were examined using immunofluorescence analysis. Also, the protein levels of Cyp17a1 and α-SMA in ovarian stromal cells were examined by western blot analysis. The expression of TGF-β1 and Smad3 signals were measured by Western blot and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis. Results: The results show that the function of ovary in POI rats were significantly improved after hUMSCs transplantation. The expression of fibrosis markers (α-SMA) and production of Collagen Type I (Collagen Ⅰ) and Collagen Type Ⅲ (Collagen Ⅲ) in POI rats were significantly inhibited in POI rats following hUMSCs transplantation. In the cultured ovarian stromal cells, the decrease of TGF-β1 and p-Smad3 protein expression was observed in hUMSCs treated POI rats. The treatment with TGF-β1 inhibitor of SB431542 further confirmed this signal pathway was involved in the process. Conclusion : Our study demonstrated that the TGF-β1 /Smad3 signaling pathway was involved in the inhibition of ovarian tissue fibrosis, which contributed to the restoration of ovarian function in POI rats following hUMSCs transplantation.

scholarly journals hUMSCs regulate the differentiation of ovarian stromal cells via  TGF-β1 /Smad3  signaling pathway to inhibit ovarian fibrosis to repair ovarian function in POI rats

2020 ◽  
Author(s):  
Linlu Cui ◽  
Hongchu Bao ◽  
Zhongfeng Liu ◽  
Xuejing Man ◽  
Hongyuan Liu ◽  
...  
Keyword(s):  
Western Blot ◽  
Signaling Pathway ◽  
Stromal Cells ◽  
Collagen Type ◽  
Ovarian Function ◽  
Ovarian Tissue ◽  
Ovarian Insufficiency ◽  
Tissue Fibrosis ◽  
Function Recovery ◽  
Tgf Β1

Abstract Objective: The basic pathological changes of primary ovarian insufficiency (POI) include ovarian tissue fibrosis and follicular development disorders. The human umbilical cord mesenchymal stem cells (hUMSCs) transplantation has been shown an effective method to improve the ovarian function in POI rat model; however, the exact mechanisms are still unclear. The purpose of this study is to investigate whether the recovery of ovarian function in POI rats is related to the inhibition of tissue fibrosis following hUMSCs transplantation. Furthermore, the transforming growth factor-β1 (TGF-β1 ) signaling pathway is explored to determine the mechanisms of ovarian function recovery through its inhibition of tissue fibrosis. Methods: The primary ovarian insufficiency (POI) rat model was established by intraperitoneal injection of chemotherapy drug cisplatin (CDDP) for 7 days, The levels of serum sex hormones were measured using enzyme-linked immunosorbent assay (ELISA). The tissue fibrosis in ovary was examined using masson staining and Sirus red staining. The collagen fibers in the ovarian tissues were detected by Western blot analysis. To investigate the mechanisms of ovarian function recovery following hUMSCs transplantation, ovarian stromal cells were isolated from the ovarian cortex of immature rats. The expression of Cytochrome P450 17A1 (Cyp17a1) and fibrosis marker of alpha smooth muscle Actin (α-SMA) in ovarian stromal cells were examined using immunofluorescence analysis. Also, the protein levels of Cyp17a1 and α-SMA in ovarian stromal cells were examined by western blot analysis. The expression of TGF-β1 and Smade3 signals were measured by Western blot and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis. Results: The results show that the function of ovary in POI rats were significantly improved after hUMSCs transplantation. The expression of fibrosis markers (α-SMA) and production of Collagen Type I (Collagen Ⅰ) and Collagen Type Ⅲ (Collagen Ⅲ) in POI rats were significantly inhibited in POI rats following hUMSCs transplantation. In the cultured ovarian stromal cells, the decrease of TGF-β1 and p-Smade3 protein expression was observed in hUMSCs treated POI rats. The treatment with TGF-β1 inhibitor of SB431542 further confirmed this signal pathway was involved in the process. Conclusion : Our study demonstrated that the TGF-β1 /Smad3 signaling pathway was involved in the inhibition of ovarian tissue fibrosis, which contributed to the restoration of ovarian function in POI rats following hUMSCs transplantation.

scholarly journals hUMSCs regulate the differentiation of ovarian stromal cells via TGF-β1/Smad3 signaling pathway to inhibit ovarian fibrosis to repair ovarian function in POI rats

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Linlu Cui ◽  
Hongchu Bao ◽  
Zhongfeng Liu ◽  
Xuejing Man ◽  
Hongyuan Liu ◽  
...  
Keyword(s):  
Western Blot ◽  
Signaling Pathway ◽  
Stromal Cells ◽  
Collagen Type ◽  
Ovarian Function ◽  
Ovarian Tissue ◽  
Primary Ovarian Insufficiency ◽  
Ovarian Insufficiency ◽  
Tissue Fibrosis ◽  
Function Recovery

Abstract Objective The basic pathological changes of primary ovarian insufficiency (POI) include ovarian tissue fibrosis and follicular development disorders. The human umbilical cord mesenchymal stem cell (hUMSC) transplantation has been shown an effective method to improve the ovarian function in POI rat model; however, the exact mechanisms are still unclear. The purpose of this study is to investigate whether the recovery of ovarian function in POI rats is related to the inhibition of tissue fibrosis following hUMSC transplantation. Furthermore, the transforming growth factor-β1 (TGF-β1) signaling pathway is explored to determine the mechanisms of ovarian function recovery through its inhibition of tissue fibrosis. Methods The primary ovarian insufficiency (POI) rat model was established by intraperitoneal injection of chemotherapy drug cisplatin (CDDP) for 7 days. The levels of serum sex hormones were measured using enzyme-linked immunosorbent assay (ELISA). The tissue fibrosis in the ovary was examined using Masson staining and Sirius red staining. The collagen fibers in the ovarian tissues were detected by Western blot analysis. To investigate the mechanisms of ovarian function recovery following hUMSC transplantation, ovarian stromal cells were isolated from the ovarian cortex of immature rats. The expression of Cytochrome P450 17A1 (Cyp17a1) and fibrosis marker of alpha smooth muscle actin (α-SMA) in ovarian stromal cells was examined using immunofluorescence analysis. Also, the protein levels of Cyp17a1 and α-SMA in ovarian stromal cells were examined by Western blot analysis. The expression of TGF-β1 and Smad3 signals was measured by Western blot and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis. Results The results show that the function of the ovary in POI rats was significantly improved after hUMSC transplantation. The expression of fibrosis markers (α-SMA) and production of Collagen Type I (Collagen I) and Collagen Type III (Collagen III) in POI rats were significantly inhibited in POI rats following hUMSC transplantation. In the cultured ovarian stromal cells, the decrease of TGF-β1 and p-Smad3 protein expression was observed in hUMSC-treated POI rats. The treatment with TGF-β1 inhibitor of SB431542 further confirmed this signal pathway was involved in the process. Conclusion Our study demonstrated that the TGF-β1/Smad3 signaling pathway was involved in the inhibition of ovarian tissue fibrosis, which contributed to the restoration of ovarian function in POI rats following hUMSC transplantation.

scholarly journals hUMSCs Regulate the Differentiation of Ovarian Stromal Cells via TGF-β1/Smad3 Signaling Pathway to Inhibit Ovarian Fibrosis to Repair Ovarian Function in POI Rats

2020 ◽  
Author(s):  
Linlu Cui ◽  
Hongchu Bao ◽  
Zhongfeng Liu ◽  
Xuejing Man ◽  
Hongyuan Liu ◽  
...  
Keyword(s):  
Western Blot ◽  
Signaling Pathway ◽  
Stromal Cells ◽  
Collagen Type ◽  
Ovarian Function ◽  
Ovarian Tissue ◽  
Primary Ovarian Insufficiency ◽  
Ovarian Insufficiency ◽  
Tissue Fibrosis ◽  
Function Recovery

Abstract Background: The basic pathological changes of primary ovarian insufficiency (POI) include ovarian tissue fibrosis and follicular development disorders. The human umbilical cord mesenchymal stem cells (hUMSCs) transplantation has been shown an effective method to improve the ovarian function in POI rat model; however, the exact mechanisms are still unclear. The purpose of this study is to investigate whether the recovery of ovarian function in POI rats are related to the inhibition of tissue fibrosis following hUMSCs transplantation. Furthermore, the transforming growth factor-β1 (TGF-β1) signaling pathway is explored to determine the mechanisms of ovarian function recovery through its inhibition of tissue fibrosis.Methods: The primary ovarian insufficiency (POI) rat model was established by intraperitoneal injection of chemotherapy drug cisplatin (CDDP) for 7 days, The levels of serum sex hormones were measured using enzyme-linked immunosorbent assay (ELISA). The tissue fibrosis in ovary was examined using masson staining and Sirus red staining. The collagen fibers in the ovarian tissues were detected by Western blot analysis. To investigate the mechanisms of ovarian function recovery following hUMSCs transplantation, ovarian stromal cells were isolated from the ovarian cortex of immature rats. The expression of Cytochrome P450 17A1 (Cyp17a1) and fibrosis marker of alpha smooth muscle Actin (α-SMA) in ovarian stromal cells were examined using immunofluorescence analysis. Also, the protein levels of Cyp17a1 and α-SMA in ovarian stromal cells were examined by western blot analysis. The expression of TGF-β1 and Smade3 signals were measured by Western blot and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis.Results: The results show that the function of ovary in POI rats were significantly improved after hUMSCs transplantation. The expression of fibrosis markers (α-SMA) and production of Collagen Type I (Collagen Ⅰ) and Collagen Type Ⅲ (Collagen Ⅲ) in POI rats was significantly inhibited in POI rats following hUMSCs transplantation. In the cultured ovarian stromal cells, the decrease of TGF-β1 and p-Smade3 protein expression was observed in hUMSCs treated POI rats. The treatment with TGF-β1 inhibitor of SB431542 further confirmed this signal pathway was involved in the process. Conclusion: Our study demonstrated that the TGF-β1/Smad3 signaling pathway was involved in the inhibition of ovarian tissue fibrosis, which contributed to the restoration of ovarian function in POI rats following hUMSCs transplantation.

scholarly journals Salidroside promoted osteogenic differentiation of adipose-derived stromal cells through Wnt/β-catenin signaling pathway

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Xiao-hua Li ◽  
Fu-ling Chen ◽  
Hong-lin Shen
Keyword(s):  
Western Blot ◽  
Osteogenic Differentiation ◽  
Signaling Pathway ◽  
Differentially Expressed Genes ◽  
Stromal Cells ◽  
Differentially Expressed ◽  
Calcium Deposition ◽  
Western Blot Assay ◽  
Adipose Derived Stromal Cells ◽  
Interfering Rna

Abstract Background Bone disease causes short-term or long-term physical pain and disability. It is necessary to explore new drug for bone-related disease. This study aimed to explore the role and mechanism of Salidroside in promoting osteogenic differentiation of adipose-derived stromal cells (ADSCs). Methods ADSCs were isolated and treated with different dose of Salidroside. Cell count kit-8 (CCK-8) assay was performed to assess the cell viability of ADSCs. Then, ALP and ARS staining were conducted to assess the early and late osteogenic capacity of ADSCs, respectively. Then, differentially expressed genes were obtained by R software. Then, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the differentially expressed genes were further analyzed. The expression of OCN, COL1A1, RUNX2, WNT3A, and β-catenin were measured by real-time PCR and Western blot analysis. Last, β-catenin was silenced by small interfering RNA. Results Salidroside significantly increased the ADSCs viability at a dose-response manner. Moreover, Salidroside enhanced osteogenic capacity of ADSCs, which are identified by enhanced ALP activity and calcium deposition. A total of 543 differentially expressed genes were identified between normal and Salidroside-treated ADSCs. Among these differentially expressed genes, 345 genes were upregulated and 198 genes were downregulated. Differentially expressed genes enriched in the Wnt/β-catenin signaling pathway. Western blot assay indicated that Salidroside enhanced the WNT3A and β-catenin expression. Silencing β-catenin partially reversed the promotion effects of Salidroside. PCR and Western blot results further confirmed these results. Conclusion Salidroside promoted osteogenic differentiation of ADSCs through Wnt/β-catenin signaling pathway.

Abstract 96: Simvastatin Prevents TGF-β1-induced SMAD2/3 Phosphorylation Involved in Ventricular Fibrosis: Role of Protein Phosphatases

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Farhan Rizvi ◽  
Ramail Siddiqui ◽  
Alessandra DeFranco ◽  
Alisher Holmuhamedov ◽  
Hao Xu ◽  
...  
Keyword(s):  
Western Blot ◽  
Cardiac Fibrosis ◽  
Collagen Type ◽  
Protein Phosphatases ◽  
Collagen Type I ◽  
Type I ◽  
Smad Protein ◽  
The Media ◽  
Ventricular Fibrosis ◽  
Tgf Β1

Background: Ventricular fibrosis leads to progressive cardiac dysfunction and heart failure (HF). Statins are reported to reduce cardiac fibrosis through the cholesterol-independent pathway, but mechanisms remain elusive. We hypothesize simvastatin reduced TGF-β1-induced ventricular fibrosis through activation of SMAD protein phosphatase Mg 2+ /Mn 2+ -1A (PPM1A), -2A (PP2A). Methods: In the absence and presence of TGF-β1 (5ng) with or without simvastatin (1μM), the rate of fibroblast proliferation (doubling time), myofibroblast differentiation (ICC), α-SMA mRNA (RT-PCR) and protein expression (Western blot) and the release of collagen synthesis markers, pro-collagen type I C-terminal peptide (PICP) and pro-collagen type III N-terminal peptide (PIIINP), in the media (ELISA) were determined along with protein interaction between SMAD2/3 and PPM1A or PP2A (Co-IP) and SMAD2/3 phosphorylation (Western blot). Results: Simvastatin reduced the effect of TGF-β1 on hVF proliferation by 47% (50000 to 26500), p<0.01; myofibroblast differentiated population from 48% (avg 48/100) to 11% (avg 11/100), p<0.01; expression of α-SMA mRNA by 76%, p<0.01; and protein by 60%, p<0.05. Simvastatin also decreased release of PICP by 66%, p<0.01, and PIIINP by 83%, p<0.01, into the media. Time-dependent increases in SMAD2/3 phosphorylation were reduced by simvastatin through activation of protein phosphatases PPM1A and PP2A by interacting with SMAD2/3. Conclusion: Involvement of PPM1A and PP2A in the anti-fibrotic effect of simvastatin reveals novel signaling mediators that may be selectively targeted for prevention of myocardial injury-induced ventricular fibrosis and HF.

Expression and immunolocalisation of follicle-stimulating hormone receptors in gonads of newborn and adult female horses

2016 ◽  
Vol 28 (9) ◽  
pp. 1340 ◽  
Author(s):  
Dragos Scarlet ◽  
Ingrid Walter ◽  
Juraj Hlavaty ◽  
Christine Aurich
Keyword(s):  
Western Blot ◽  
Granulosa Cells ◽  
Hormone Receptors ◽  
Ovarian Function ◽  
Ovarian Tissue ◽  
Cumulus Cells ◽  
Protein Levels ◽  
Fshr Gene ◽  
Luteal Tissue ◽  
Immunofluorescence Labelling

In mares, FSH and its receptor (FSHR) are essential for ovarian function. The objective of the present study was to analyse FSHR gene expression at the mRNA and protein levels in ovarian tissue from newborn and adult horses. Expression of mRNA was analysed by reverse transcription polymerase chain reaction, whereas FSHR protein was visualised by immunohistochemistry (IHC), immunofluorescence labelling (IF) and western blot. FSHR mRNA was detected in ovarian follicles and luteal tissue from adult mares, as well as in the ovaries of neonates. Follicular growth up to 4 mm in diameter was already present in neonates. Using IHC and IF, FSHR protein was detected in granulosa cells, cumulus cells and inconsistently in oocytes, independent of the animal’s age or the stage of folliculogenesis. A lower FSHR expression was observed in theca cells in comparison to granulosa cells. FSHR was abundant in the ovarian stroma cells of neonates but not of adults. Luteal cells stained positive for FSHR independent of the stage of corpus luteum development. The presence of FSHR protein in various cell populations of the ovary was confirmed by western blot. In conclusion, FSHR is present in horse ovaries consistently from birth onwards and expression remains constant during the oestrous cycle.

scholarly journals FERTILITY PRESERVATION: Freezing of ovarian tissue and clinical opportunities

Reproduction ◽  
2019 ◽  
Vol 158 (5) ◽  
pp. F27-F34 ◽  
Author(s):  
C Yding Andersen ◽  
L S Mamsen ◽  
S G Kristensen
Keyword(s):  
Fertility Preservation ◽  
Ovarian Function ◽  
Ovarian Tissue ◽  
Genetic Diseases ◽  
Endocrine Function ◽  
Ovarian Tissue Cryopreservation ◽  
Ovarian Insufficiency ◽  
Technical Aspects ◽  
Increased Risk ◽  
Tissue Cryopreservation

Ovarian tissue cryopreservation (OTC) is mainly used for fertility preservation in girls and women facing a gonadotoxic treatment. If the woman subsequently becomes menopausal, the ovarian tissue may be transplanted to regain ovarian function, including fertility. The method was developed more than two decades ago and today thousands of women worldwide have undergone OTC. Fewer than 500 patients have had tissue transplanted and close to 100% of those regain ovarian function. Several technical aspects of OTC are now becoming more established, including high quantitative follicle survival, defining the size of the tissue resulting in optimal tissue revascularisation and follicle loss resulting from transport of ovarian tissue prior to freezing. We have used OTC to safeguard fertility in patients with genetic diseases, which for some diagnoses is purely experimental, as no transplantations is yet been performed. Usage of OTC beyond fertility is now also being considered; here, the endocrine function of follicles is the focus. It has been suggested that ovarian tissue stored in the reproductive years may be used to avoid premature ovarian insufficiency (POI) when there is a familial disposition or to postpone menopause in patients with an increased risk of osteoporosis or cardiovascular diseases. The benefit of OTC beyond fertility requires, however, actual clinical studies. The current review includes several recent technical aspects with contributions from Denmark building on some of the early work by Roger Gosden.

scholarly journals Schisantherin A Inhibits Pulmonary Fibrosis via Regulating ERK Signaling Pathway

2020 ◽  
Vol 15 (8) ◽  
pp. 1934578X2094835
Author(s):  
Wenyue Zhuang ◽  
Na Zhao ◽  
Di Li ◽  
Xiaoming Su ◽  
Yueyang Wang ◽  
...  
Keyword(s):  
Pulmonary Fibrosis ◽  
Western Blot ◽  
Signaling Pathway ◽  
Inflammatory Cytokines ◽  
Erk Signaling ◽  
Mesenchymal Transition ◽  
Tgf Β1 ◽  
Schisantherin A

There is no effective method for treating pulmonary fibrosis (PF) until now. This study investigated the anti-fibrotic effect of schisantherin A (SCA) extracted from Schisandra chinensis and its potential molecular mechanism in PF. A bleomycin-induced PF mouse model in vivo and transforming growth factor (TGF)-β1-induced A549 epithelial-mesenchymal transition (EMT) cell model in vitro were used for assessing the anti-fibrotic effect of SCA. Histopathological examination was conducted after hematoxylin and eosin and Masson staining. The level of TGF-β1 was tested by ELISA. The expression levels of α-smooth muscle actin, E-cadherin, and inflammatory cytokines (COX2, IL-1β, IL-6, and TNF-α) were determined by quantitative reverse transcription polymerase chain reaction and Western blot. The expression of extracellular signal-regulated kinase (ERK) was tested in lung tissues and cells by Western blot. The in vivo experiments revealed that SCA treatment markedly improved body weight and pulmonary index and reformed the destruction of the lung tissue structure. We observed that SCA inhibited the process of TGF-β1-induced EMT in the in vitro experiments. Inflammatory cytokines were reduced greatly in lung tissues and cells by SCA. Our study also indicated that SCA decreased phosphorylated ERK. It was concluded that SCA can attenuate PF by regulating the ERK signaling pathway, which suggests that SCA may be used as a potential therapeutic drug for PF.

scholarly journals MO014DANHONG INJECTION INHIBITS LIPOPOLYSACCHARIDE-ENHANCED CELL PROLIFERATION OF RAT RENAL MESANGIAL CELLS VIA NF-ΚB SIGNALING PATHWAY

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
Liu Hua ◽  
Lei Chen ◽  
Limin Wei ◽  
Hongli Jiang
Keyword(s):  
Cell Proliferation ◽  
Protein Expression ◽  
Western Blot ◽  
Signaling Pathway ◽  
Inflammatory Mediators ◽  
Mesangial Cells ◽  
Inhibitory Effect ◽  
Cell Counting ◽  
Pyrrolidine Dithiocarbamate ◽  
Tgf Β1

Abstract Background and Aims Mesangioproliferative glomerulonephritis (MsPGN) is well known as one of the leading causes of end-stage renal failure (ESRD), especially in the Asian population. The principal characteristic of MsPGN is the over proliferation of glomerular mesangial cells (MCs) accompanied by expansion of the extracellular matrix (ECM). Our previous studies demonstrated that Danhong injection (DHI), is a traditional Chinese medicine injection, could improve the renal function and inhibit the MCs proliferation and ECM expansion in rats with MsPGN. However, the molecular mechanisms have not been fully elucidated. To explore the potential mechanisms of DHI in the treatment of MsPGN, we investigated the effects of DHI on lipopolysaccharide (LPS)-induced nuclear factor-kappa B (NF-κB) activation and its downstream inflammatory mediators, such as intercellular adhesion molecule-1 (ICAM-1), transforming growth factor-beta 1 (TGF-β1), inducible nitric oxide synthase (iNOS) and fibronectin (FN) protein expression in rat MCs. Method The rat MCs treated with different concentrations of DHI (0, 50, 100, 200, 500, 1000, and 2000 uL/L) for 12 h, then incubated with or without 100 ng/ml LPS for another 24 h. Subsequently, cell proliferation was determined by Cell Counting Kit-8 (CCK8). Furthermore, the rat MCs treated with low-dose DHI (250 uL/L), median-dose DHI (500 uL/L) and high-dose DHI (1000 uL/L) for 12 h or Ammonium pyrrolidine dithiocarbamate (PDTC) for 30 min before 24h treatment of LPS. Then the activation of NF-κB was detected by Western blot and immunofluorescence. The protein levels of ICAM-1, TGF-β1, iNOS and FN in rat MCs were detected by Western blot. Results The results of CCK-8 revealed that DHI significantly suppressed LPS-induced cell proliferation (shown in Fig.1). LPS stimulation resulted in a significant increment of p65 contents in nucleus and a decrement of p65 contents in cytoplasm in rat MCs compared with NC. PDTC and DHI exerted potent inhibitory effect on increasing expression of p65 in nucleus and decreasing in cytoplasm compared with LPS treatment group. The inhibitory effect on NF-κB nuclear translocation of DHI was in a dose- dependent manner (shown in Fig.2). The Western blot assay showed that the protein level of IκB-α in cytoplasm treated by LPS decreased significantly compared with that in control (shown in Fig. 3) and this decrement was significantly reversed by PDTC and DHI. In addition, the protein expression of ICAM-1, TGF-β1, iNOS and FN was also inhibited by PDTC and DHI (shown in Fig. 4). Conclusion DHI significantly repressed LPS-induced cell proliferation and FN expression in rat MCs through inhibiting the activation of NF-κB signaling pathway and protein expression of its downstream inflammatory mediators. The ameliorative effects of DHI on MsPGN might be associated with this inhibition effect on NF-B signaling pathway.

Study on Receptor-Regulated Smad Protein of Acute Promyelocytic Leukemia Cell Differentiation Process in TGF-β1 Signaling Pathway.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4078-4078
Author(s):  
Jiayao Lin ◽  
Fangyuan Chen ◽  
Hua Zhong ◽  
Hairong Wang ◽  
Renrong Ouyang ◽  
...  
Keyword(s):  
Protein Expression ◽  
Western Blot ◽  
Signaling Pathway ◽  
Transcriptional Activity ◽  
Control Groups ◽  
Nb4 Cells ◽  
The Third ◽  
Realtime Pcr ◽  
Smad3 Protein ◽  
Tgf Β1

Abstract It was reported that several members of TGF-β1 signaling pathway had the situation of reduced expression, absence or abnormal function in mononuclear cell of acute leukemia patients. Our preliminary experiments also found that the expression of TGF-β1 receptors and several downstream Smad proteins decreased considerably than those after induced differentiation in a APL cell line (NB4) and primarily cells of APL patients. In order to further investigate the important links in TGF-β1 pathway in induced differentiation of APL cells by ATRA, R-Smad, namely the change of Smad2 and Smad3 protein expression, we detected mRNA and the protein expression of Smad2,Smad3 by Realtime-PCR and Western blot after we brought ATRA to act on cell NB4 and primary cells of patient for 0, 3, 6, 12, 24, 48, 72 and 96 hours. Cells treated with dehydrated alcohol were harvested at the given time as control group. As to NB4, The expression of Smad2 and Smad3 mRNA tested showed a significant increase treated with ATRA measured by PCR. In contrast, the expression levels in the control groups remained almost unchanged with time(p<0.01). After ATRA induced, Smad2 and Smad3 levels emerged a gradual rising trend, reached maximum value up to 48h (126.11±1.06,7780.09±288.58)and then gradually declined. Western blots showed that the degree of Smad2 protein was increased at 3h, which peaked at 48h, before returning to baseline level at 72h. Simultaneously, elevation of Smad3 was observed. The expression level was increased within 6h, up to the peak at 72h which was later than Smad2, and then dropped down. In the aspect of the primary cells, Western results showed that three De novo patients had extremely low Smad2 and Smad3 protein expression, but after activated by ATRA for 48h, their levels were increased significantly. These experiment results showed that a R-Smad reduction or absence might exist in APL, and that APL cells differentiation might something to do with up-regulating of expression of R-Smad. Its concrete mechanism might be associated with PML-RARα fusion protein. We analyzed the influence of PML-RARα on the transcriptional activity of the (CACA) 9-lux reporter gene plasmid(including nine tandem repeated CAGA sequence that may combine Smad3 and Smad4) and found that PML-RARα may inhibit the transcription regulation of R-Smad. In IF experiment, we observed that there was a phenomenon that PML-RARa protein in NB4 cells combined with Smad2, Smad3 protein respectively. The phenomenon presented an obvious falling tendency after activated by ATRA for 24, 48 and 72 hours. HK Lin et al. found that cytoplasmic PML (cPML) in normal cells could mediate the formation of complex TGF-β1 receptor/R-Smad/SARA, and thus activate TGF-β1 signaling pathway. We guess that PML-RARa may combine with the R-Smad, therefore, block the transcription control for genes of TGF-β1 pathway. Figure 1. The expression of R-Smad mRNA and protein in ATRA treated NB4 cells. The cells were exposed to ATRA (experimental groups) or not (control groups). At given time (0h, 3h, 6h, 12h, 24h, 48h, 72h, 96h) cells were harvest and subjected to Realtime-PCR and western blot Figure 1. The expression of R-Smad mRNA and protein in ATRA treated NB4 cells. The cells were exposed to ATRA (experimental groups) or not (control groups). At given time (0h, 3h, 6h, 12h, 24h, 48h, 72h, 96h) cells were harvest and subjected to Realtime-PCR and western blot Figure 2. The influence of PML-RARα fusion protien on the transcriptional activity of the (CACA) 9-lux reporter gene plasmid in NB4 cells. In the first group, with plasmid (CACA) 9-lux (nine Smad3 repeat sequences included). In the second group, with (CACA) 9-lux and empty vector, and in the third group, with (CACA) 9-lux and plasmid PML-RARα. The transcriptional activity of the third group was lower than the other two groups. (p<0.05) Figure 2. The influence of PML-RARα fusion protien on the transcriptional activity of the (CACA) 9-lux reporter gene plasmid in NB4 cells. In the first group, with plasmid (CACA) 9-lux (nine Smad3 repeat sequences included). In the second group, with (CACA) 9-lux and empty vector, and in the third group, with (CACA) 9-lux and plasmid PML-RARα. The transcriptional activity of the third group was lower than the other two groups. (p<0.05)

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