Gene Expression Levels Of Collagen Research Articles

Comparison of ligamentization potential between anterior cruciate ligament-derived cells and adipose-derived mesenchymal stem cells reseeded to acellularized tendon allograft.

To test the hypothesis that reseeded anterior cruciate ligament (ACL)-derived cells have a better ability to survive and integrate into tendon extracellular matrix (ECM) and accelerate the ligamentization process, compared to adipose-derived mesenchymal stem cells (ADMSCs). Acellularized tibialis allograft tendons were used. Tendons were randomly reseeded with ACL-derived cells or ADMSCs. ACL-derived cells were harvested and isolated from remnants of ruptured ACLs during reconstruction surgery and cultured at passage three. Cell suspensions (200 µl) containing 2 × 106 ACL-derived cells or ADMSCs were prepared for the purpose of reseeding. At days 1, 3, and 7 post-reseeding, graft composites were assessed for repopulation with histological and immunohistochemical analysis. Matrix protein contents and gene expression levels were analyzed. In the graft reseeded with ACL-derived cells, a large number of elongated cells that integrated into the matrix were evident at day 3 and day 7. However, in the graft reseeded with ADMSCs, only a small number of elongated cells were found integrated into the matrix. Immunofluorescence for Ki-67 and type I collagen confirmed the pronounced production of type I collagen by Ki-67-positive ACL-derived cells integrated into the ECM. A messenger RNA (mRNA) expression assay demonstrated significantly higher gene expression levels of types I (p = 0.013) and III (p = 0.050) collagen in the composites reseeded with ACL-derived cells than ADMSCs. ACL-derived cells, when reseeded to acellularized tendon graft, demonstrated earlier better survival and integration in the tendon ECM and resulted in higher gene expression levels of collagen, which may be essential to the normal ligamentization process compared to ADMSCs.Cite this article: Bone Joint Res2022;11(11):777-786.

Berberine Encapsulated in Exosomes Derived from Platelet-Rich Plasma Promotes Chondrogenic Differentiation of the Bone Marrow Mesenchymal Stem Cells via the Wnt/β-Catenin Pathway.

Cartilage regenerative medicine, wherein the stem cells from adults exert a crucial role, has high potential in the treatment of defective articular cartilage. Recently, Bone marrow mesenchymal stem cells (BMSCs) are being increasingly recognized as an alternative source of adult stem cells, which are capable of differentiating into several cell types (e.g., adipocytes, chondrocytes, and osteoblasts). However, their proliferative properties and tendency to dedifferentiate restrict their use in clinical settings. Recently, a possible bioactive material PRP-exos (exosomes derived from platelet-rich plasma), has emerged, which can effectively facilitate the differentiation and proliferation of cells. Recent studies have reported that berberine (Ber), known to have anti-inflammatory properties, plays a role in osteogenesis. Since biological molecules are used in combinations, we attempted to assess the effect of Exos-Ber (PRP-exos in combination with Ber) on the chondrogenic differentiation of BMSCs in vitro. In this study, Exos-Ber was observed to promote the proliferation of BMSCs and cause their chondrogenic differentiation in vitro. Additionally, Exos-Ber could promote the migration of BMSCs and increase the protein expression of the chondrogenic genes (Collagen II, SOX9, Aggrecan). After treatment with Exos-Ber, significant induction of β-catenin expression was observed, which could be repressed successfully by adding β-catenin inhibitor XAV-939. Interestingly, the repression of the Wnt/β-catenin axis also resulted in reduced gene expression levels of Collagen II, SOX9, and Aggrecan. These observations indicated that Exos-Ber facilitated the differentiation of chondrogenic BMSCs by modulating the Wnt/β-catenin axis, which offers innovative insights into the reconstruction of cartilage.

Lactoferrin with Zn-ion protects and recovers fibroblast from H2O2-induced oxidative damage

Lactoferrin (LF) has attracted great attention due to its various bioactivities, which depend on the degree of saturation with different cations. This study focused on the synergistic effect of LF and Zn2+ on human gingival fibroblasts (hGFs), considering antioxidant activities, cell proliferation, and collagen gene expression levels in these cells to improve the wound healing. The hGFs were cultured in an experimental medium, containing 1000 μg/mL of LF and various concentrations of ZnCl2. The cells were subjected to oxidative damage by exposure to 600 μM H2O2 for 30 min before incubation in the experimental medium. The cell proliferation rate and the relative gene expression levels of genes associated with apoptosis, antioxidant enzymes, and collagen were compared. H2O2 decomposition by LF was also measured using a colorimetric assay. LF enhanced hGF proliferation and the expression of collagen. Furthermore, LF directly scavenged H2O2 and prevented lipid peroxidation by enhancing the expression of glutathione peroxidase 4 gene expression, resulting in the prevention of apoptosis and recovery of the cells from H2O2-induced oxidative damage. The addition of ZnCl2 enhanced these results. The results indicated that LF with Zn-ion could play an important role in modulating the functions related to wound healing.

Effects of low-density pulsed ultrasound treatment on transforming growth factor-beta, collagen level, histology, biomechanics, and function in repaired rat tendons.

Objectives The aim of this study is to compare the effects of low-density pulsed ultrasound (LIPUS) treatment on growth factors/collagen production, histological, biomechanical, and function of rats with Achilles tendon injury. Materials and methods A total of 44 Wistar Albino rats were used in the study between April 2017 and June 2018. The rats were randomized to two treatment groups. Group 1 (n=6) received LIPUS treatment (0.3 Watt/cm2; 1 MHz, 1:5 pulse mode) and Group 2 (n=6)received sham ultrasound (US) treatment following Achilles tendon surgery. Transforming growth factor-beta 1 (TGF-β1) and collagen gene expression levels were evaluated using polymerase chain reaction. The histological evaluation was performed with the Bonar scoring system. The tensile strength was measured by biomechanical testing and the function was evaluated with the Achilles Functional Index (AFI). Results Although TGF-β1 expression and tensile strength evaluation showed a tendency to improve in favor of the LIPUS group, no statistically significant difference was found (p=0.065 and p=0.053, respectively). The COL3 gene expression in the LIPUS group and the COL1 expression in the sham US group were significantly higher. Bonar scores and AFI scores showed a statistically significant improvement in the LIPUS group, compared to the sham US group. Conclusion Our study results show that LIPUS yields positive effects on tendon histology and functional status in repaired Achilles tendon in rats.

Effects of Ethanol Feeding in Early-Stage NAFLD Mice Induced by Western Diet

Background: The prevalence of metabolic liver diseases is increasing and approved pharmacological treatments are still missing. Many animal models of nonalcoholic fatty liver disease (NAFLD) show a full spectrum of fibrosis, inflammation and steatosis, which does not reflect the human situation since only up to one third of the patients develop fibrosis and nonalcoholic steatohepatitis (NASH). Methods: Seven week old C57Bl/J mice were treated with ethanol, Western diet (WD) or both. The animals’ liver phenotypes were determined through histology, immunohistochemistry, Western blotting, hepatic triglyceride content and gene expression levels. In a human cohort of 80 patients stratified by current alcohol misuse and body mass index, liver histology and gene expression analysis were performed. Results: WD diet and ethanol-treated animals showed severe steatosis, with high hepatic triglyceride content and upregulation of fatty acid synthesis. Mild fibrosis was revealed using Sirius-red stains and gene expression levels of collagen. Inflammation was detected using histology, immunohistochemistry and upregulation of proinflammatory genes. The human cohort of obese drinkers showed similar upregulation in genes related to steatosis, fibrosis and inflammation. Conclusions: We provide a novel murine model for early-stage fatty liver disease suitable for drug testing and investigation of pathophysiology.

Effects of Experimental Hyperthyroidism on Collagen IV and Laminin-α5 Gene Expression in Balb/C Mouse Seminiferous Tubules

Background: Hyperthyroidism is one of the disorders of the thyroid gland, an organ that controls the cellular and molecular behaviors of the seminiferous tubule basement membrane (BM), and ultimately, influences its evolutionary process. We aimed to investigate the effects of hyperthyroidism on immunohistochemical characteristics and gene expression levels of collagen IV and laminin-α5 in seminiferous tubules BM of Balb/C mice. Materials and Methods: Twenty male Balb/C mice were divided into experimental and control groups. The experimental group received 500 mg/l of levothyroxine (L-thyroxine) diluted in drinking water for two months to inducing hyperthyroidism, which was confirmed by radioimmunoassay. At the end of the study, the mice were sacrificed, and their testes were extracted for immunohistochemistry and real-time polymerase chain reaction assays. Results: Although a weak reaction was observed in the experimental specimens, no significant enhancement was noted in color intensity of type IV collagen in the seminiferous tubules BM of the experimental group as compared to the control group (P>0.05). Collagen IV gene expression results in the experimental group were not significantly different from the controls (P>0.05). Thus, there was a significant increase in laminin α5 gene expression compared to the control group (P=0.016). Conclusion: Considering the key role of collagen IV and laminin-α5 in the seminiferous tubule BM in the testes, the results of this study indicated that hyperthyroidism has important effects on both structures and functions of these two components. [GMJ.2019;8:e1369]

Effects of Experimental Hyperthyroidism on Collagen IV and Laminin-α5 Gene Expression in Balb/C Mouse Seminiferous Tubules.

Background: Hyperthyroidism is one of the disorders of the thyroid gland, an organ that controls the cellular and molecular behaviors of the seminiferous tubule basement membrane (BM), and ultimately, influences its evolutionary process. We aimed to investigate the effects of hyperthyroidism on immunohistochemical characteristics and gene expression levels of collagen IV and laminin-α5 in seminiferous tubules BM of Balb/C mice. Materials and Methods: Twenty male Balb/C mice were divided into experimental and control groups. The experimental group received 500 mg/l of levothyroxine (L-thyroxine) diluted in drinking water for two months to inducing hyperthyroidism, which was confirmed by radioimmunoassay. At the end of the study, the mice were sacrificed, and their testes were extracted for immunohistochemistry and real-time polymerase chain reaction assays. Results: Although a weak reaction was observed in the experimental specimens, no significant enhancement was noted in color intensity of type IV collagen in the seminiferous tubules BM of the experimental group as compared to the control group (P>0.05). Collagen IV gene expression results in the experimental group were not significantly different from the controls (P>0.05). Thus, there was a significant increase in laminin α5 gene expression compared to the control group (P=0.016). Conclusion: Considering the key role of collagen IV and laminin-α5 in the seminiferous tubule BM in the testes, the results of this study indicated that hyperthyroidism has important effects on both structures and functions of these two components.

Stem Cells Isolated from the Sheath and Core Tissues of Human Plantar Fascia Exhibit Differential Biological Properties

Category: Hindfoot Introduction/Purpose: Plantar fasciitis is a very common ligament disease which affects more than 3 million patients in the United States every year. However, there is no effective treatment in clinics because its pathogenesis is largely unknown. Although PF has been studied quite well from a biomechanical viewpoint, little has been done on PF biology. To better understand the cellular mechanisms of plantar fasciitis, in this study we aimed first to characterize biological properties of stem cells isolated from the sheath and the core of human PF tissues. Their biological responses to mechanical loading are also studied. Methods: Two stem cell populations isolated from the sheath (PF-S) and core (PF-C) parts of human PF tissues were investigated by characterizing their colony formation, stem cell marker expression, population doubling time (PDT), multi-differentiation potentials, and biological responses to mechanical loading. One-way ANOVA followed by Fisher’s PLSD for multiple comparisons was used for statistical analysis. A p-value < 0.05 was considered to be statistically significant. Results: PF-S grew quicker and formed larger colonies than PF-C cells (Fig. 1). More PF-S expressed nucleostemin (Fig. 2A), Oct- 4 (Fig. 2C), SSEA-4 (Fig. 2E) than PF-C cells (Fig. 2B, 2D, 2F). More PF-S were stained by collagen type IV (Fig. 3D), CD31 (Fig. 3A), and vimentin (Fig. 3B), and less stained by collagen I (Fig. 3C) than those of PF-C cells (Fig. 3E-I). More PF-S were differentiated into adipocytes (Fig. 4A), osteocytes (Fig. 4B), and chondrocytes (Fig. 4C) than PF-C cells (Fig. 4D-F). PF-S and PF-C respond to intensive mechanical stretching differentially in terms of gene expression levels of collagen I (Fig. 5A), IV (Fig. 5B), CD105 (Fig. 5C), non-tenocyte related genes (Fig. 5D-F), MMPs (Fig. 5G-H), and inflammation-related genes, COX-I and COX-II (Fig. 5I-J). Conclusion: This study characterized plantar fascia in detail at histological, cellular, and mechanobiological levels. Our results showed that plantar fascia consists of two distinct tissues sheath and core with differential structural and cellular properties. The cells isolated from sheath and core of human PF also differ in their response to mechanical loading. This information may be useful in determining the respective contributions of the sheath and core stem cells to the development of plantar fasciitis and in devising new biologic approaches for effective repair of injured PF in clinics.

Icariin promotes stable chondrogenic differentiation of bone marrow mesenchymal stem cells in self‑assembling peptide nanofiber hydrogel scaffolds.

Icariin, a traditional Chinese medicine, has previously been demonstrated to promote chondrogenesis of bone marrow mesenchymal stem cells (BMSCs) in traditional 2D cell culture. The present study investigated whether icariin has the potential to promote stable chondrogenic differentiation of BMSCs without hypertrophy in a 3D microenvironment. BMSCs were cultivated in a self-assembling peptide nanofiber hydrogel scaffold in chondrogenic medium for 3 weeks. Icariin was added to the medium throughout the culture period at concentrations of 1×10−6 M. Chondrogenic differentiation markers, including collagen II and SRY-type high mobility group box 9 (SOX9) were detected by immunofluorescence, reverse transcription-quantitative polymerase chain reaction and toluidine blue staining. Hypertrophic differentiation was further assessed by detecting collagen X and collagen I gene expression levels and alkaline phosphatase activity. The results demonstrated that icariin significantly enhanced cartilage extracellular matrix synthesis and gene expression levels of collagen II and SOX9, and additionally promoted more chondrocyte-like rounded morphology in BMSCs. Furthermore, chondrogenic medium led to hypertrophic differentiation via upregulation of collagen X and collagen I gene expression levels and alkaline phosphatase activity, which was not potentiated by icariin. In conclusion, these results suggested that icariin treatment may promote chondrogenic differentiation of BMSCs, and inhibit the side effect of growth factor activity, thus preventing further hypertrophic differentiation. Therefore, icariin may be a potential compound for cartilage tissue engineering.

697 Effects of Gadodiamide on cell proliferation and collagen production in cultured human dermal fibroblasts

Nephrogenic systemic fibrosis (NSF) is a disease characterized by fibrosis of the systemic organs in patients with renal failure. Following the findings of recent epidemiological studies and the finding of gadolinium (Gd) in the skin tissue of NSF patients, it is now definitely known that the use of Gd contrast agents can trigger NSF. To date, however, the exact mechanism underlying the induction of fibrosis in various organs by Gd remains unexplained. The present study was undertaken to evaluate the influence of Gd on the proliferation activity and collagen production of cultured fibroblasts. Normal human dermis-derived fibroblasts were incubated in the presence of gadodiamide (GA). The proliferation activity of the cells was assessed on the basis of the cell counts in the fibroblast growth curve and the DNA-synthetic activity of the cells (indicator; level of 3H-thymidine uptake by cells). The collagen production was evaluated by densitometric measurement of the quantity of collagen through electrophoresis and fluorography after incorporation of 3H-proline into the procollagens. Furthermore, the expression levels of the genes for type I and III collagen were measured by real-time reverse transcription polymerase chain reaction (RT-PCR) assay. The cell count tended to be higher when the fibroblasts were incubated in medium containing GA, furthermore, the DNA synthetic activity also rose in a concentration-dependent manenr in the GA-treated group as compared to that in the control group. No significant changes in either the collagen production or the collagen gene expression levels were noted in cultures containing GA. These results suggest that the formation of sclerosing lesions in patients with NSF may be attributable to the effect of GA of enhancing the growth activity of fibroblasts.

Tissue alignment enhances remodeling potential of tendon-derived cells - Lessons from a novel microtissue model of tendon scarring

Tendinopathy is a widespread and unresolved clinical challenge, in which associated pain and hampered mobility present a major cause for work-related disability. Tendinopathy associates with a change from a healthy tissue with aligned extracellular matrix (ECM) and highly polarized cells that are connected head-to-tail, towards a diseased tissue with a disorganized ECM and randomly distributed cells, scar-like features that are commonly attributed to poor innate regenerative capacity of the tissue. A fundamental clinical dilemma with this scarring process is whether treatment strategies should focus on healing the affected (disorganized) tissue or strengthen the remaining healthy (anisotropic) tissue. The question was thus asked whether the intrinsic remodeling capacity of tendon-derived cells depends on the organization of the 3D extracellular matrix (isotropic vs anisotropic). Progress in this field is hampered by the lack of suitable in vitro tissue platforms. We aimed at filling this critical gap by creating and exploiting a next generation tissue platform that mimics aspects of the tendon scarring process; cellular response to a gradient in tissue organization from isotropic (scarred/non-aligned) to highly anisotropic (unscarred/aligned) was studied, as was a transient change from isotropic towards highly anisotropic. Strikingly, cells residing in an ‘unscarred’ anisotropic tissue indicated superior remodeling capacity (increased gene expression levels of collagen, matrix metalloproteinases MMPs, tissue inhibitors of MMPs), when compared to their ‘scarred’ isotropic counterparts. A numerical model then supported the hypothesis that cellular remodeling capacity may correlate to cellular alignment strength. This in turn may have improved cellular communication, and could thus relate to the more pronounced connexin43 gap junctions observed in anisotropic tissues. In conclusion, increased tissue anisotropy was observed to enhance the cellular potential for functional remodeling of the matrix. This may explain the poor regenerative capacity of tenocytes in chronic tendinopathy, where the pathological process has resulted in ECM disorganization. Additionally, it lends support to treatment strategies that focus on strengthening the remaining healthy tissue, rather than regenerating scarred tissue.

Hyaluronic Acid Coating Enhances Biocompatibility of Nonwoven PGA Scaffold and Cartilage Formation.

Synthetic polymers such as polyglycolic acid (PGA) fibers are the traditional tissue engineering scaffolds that are widely used for engineering a variety of soft tissues. However, the major disadvantage of this polymer material is its released acidic degradation products that trigger inflammatory response and fibrotic process, which affects the biocompatibility and the quality of the engineered tissues. In this study, the effect of hyaluronic acid (HA) coating on improving PGA biocompatibility was explored. The results showed that 1% HA solution could better coat PGA fibers than other tested concentrations of HA, and coated PGA exhibited less inflammatory reaction upon in vivo subcutaneous implantation. In vitro characterization demonstrated that HA coating could enhance cell adhesion to the scaffold and reduce gene expression of IL-1, IL-6, IL-8, and α-SMA. It also decreased the acidity of degradation products in vitro. Furthermore, coated PGA could engineer better cartilages in vitro with higher content of total collagen and glycosaminoglycan, as well as higher gene expression levels of collagen II, aggrecan, and Sox9. Collectively, the data indicate that HA coating can significantly enhance the biocompatibility of this traditional scaffold material, which also enhances the quality of engineered tissues.

Effects of Gadodiamide on cell proliferation and collagen production in cultured human dermal fibroblasts.

Nephrogenic systemic fibrosis (NSF) is a disease characterized by fibrosis of the systemic organs in patients with renal failure. Following the findings of recent epidemiological studies and the finding of gadolinium (Gd) in the skin tissue of NSF patients, it is now definitely known that the use of Gd contrast agents can trigger NSF. To date, however, the exact mechanism underlying the induction of fibrosis in various organs by Gd remains unexplained. This study was undertaken to evaluate the influence of Gd on the proliferation activity and collagen production of cultured fibroblasts. Normal human dermis-derived fibroblasts were incubated in the presence of gadodiamide (GA) in the concentration range of 5×10-7 to 5×10-3 M. The proliferation activity of the cells was assessed on the basis of the cell counts in the fibroblast growth curve and the DNA-synthetic activity of the cells (indicator; level of 3H-thymidine uptake by cells). The collagen production was evaluated by densitometric measurement of the quantity of collagen through electrophoresis and fluorography after incorporation of 3H-proline into the procollagens. Furthermore, the expression levels of the genes for type I and III collagen were measured by real-time reverse transcription polymerase chain reaction (RT-PCR) assay. The cell count tended to be higher when the fibroblasts were incubated in medium containing GA in the concentration range of 5×10-7 to 5×10-4M as compared to that in the GA-free control cultures; furthermore, the DNA-synthetic activity also rose in a concentration-dependent manner in the GA-treated group as compared to that in the control group. No significant changes in either the collagen production or the collagen gene expression levels were noted in cultures containing GA at concentrations between 5×10-7 and 5×10-3 M. These results suggest that the formation of sclerosing lesions in patients with NSF may be attributable to the effect of GA of enhancing the growth activity of fibroblasts.

Partial deletion of the ROCK2 protein fails to reduce renal fibrosis in a unilateral ureteral obstruction model in mice.

Renal fibrosis is a well‑known cause for the progression of chronic kidney disease. Rho/Rho‑associated coiled‑coil kinase (ROCK) signaling is involved in renal fibrotic processes. Non‑selective ROCK1/2 inhibitors have been reported to reduce renal interstitial fibrosis in a rodent unilateral ureteral obstruction (UUO) model. To clarify the role and contribution of ROCK2 in renal fibrosis, the present study used ROCK2 heterozygous knockout (HKO) mice to assess collagen deposition and fibrosis‑associated gene expression in the kidney of the UUO model. In the ROCK2 HKO mice, the expression level of ROCK2 in the normal kidney was half of that in the kidney of wild‑type (WT) mice. The expression levels of ROCK1 in the ROCK2 HKO mice and WT mice were equivalent. Furthermore, in the ROCK2 HKO and the WT mice, the hydroxyproline content and the gene expression levels of collagen I and transforming growth factor‑β1 in the obstructed kidneys were augmented following UUO. By contrast, the mRNA expression of α‑smooth muscle actin decreased in the ROCK2 HKO mice, compared with that in the WT mice. The activity of ROCK in the obstructed kidneys, indicated by the phosphorylation of myosin phosphatase target subunit‑1, which is a non‑selective substrate of ROCK1 and ROCK2, was equivalent among the ROCK2 HKO and WT mice. In conclusion, no differences in renal interstitial fibrosis or UUO‑induced ROCK activity were identified between the ROCK2 HKO and WT mice, indicating that the genetic partial disruption of ROCK2 is insufficient for protecting against renal fibrosis.

The effects of different molecular weight chondroitin-4-sulfates in chondrocyte pellet culture.

For this study, we cultured chondrocyte pellets in Dulbecco's modified Eagle's medium plus a 2% fetal bovine serum medium, and treated them with 2- to 8-mer oligosaccharides of chondroitin sulfate A to examine the effects of these oligosaccharides on the differentiation and protection of chondrocytes. We found low-molecular-weight CSAs to increase the ratio of the gene expression levels of collagen II/collagen I of chondrocytes from the first day up to 14days after culture compared with those under a CSA-free medium. Moreover, low-molecular-weight CSAs inhibited the expression of matrix metalloproteinases and peptidases, and stimulated an endogenous tissue inhibitor of metalloproteinases. The dp-8 (8-mer) CSA yielded the most effective response among promoting collagen type II protein secretions compared with other groups.

Chymase inhibitor ameliorates hepatic steatosis and fibrosis on established non‐alcoholic steatohepatitis in hamsters fed a methionine‐ and choline‐deficient diet

Chymase plays a role in the augmentation of angiotensin II formation, which is involved in liver fibrosis. The therapeutic effects of a chymase inhibitor, TY-51469, on established hepatic steatosis and fibrosis were investigated in a model of developed non-alcoholic steatohepatitis. Hamsters were fed a normal diet or methionine- and choline-deficient (MCD) diet for 12 weeks. Then, treatment with TY-51469 (1 mg/kg per day) or placebo was initiated, and the treatment was continued concurrently with the MCD diet for an additional 12 weeks. At 12 weeks after initiating the MCD diet, marked hepatic steatosis and fibrosis were observed in MCD diet-fed hamsters. Malondialdehyde and gene expression levels of collagen I, collagen III, α-smooth muscle actin (α-SMA) and Rac-1 in liver extracts were also increased in the MCD-diet-fed hamsters at 12 weeks. At 24 weeks, hepatic steatosis and fibrosis were more prominent in the placebo-treated hamsters that were fed the MCD-diet for 24 weeks versus 12 weeks. Hamsters treated with TY-51469 for 12 weeks after being on a 12-week MCD diet had significant ameliorations in both hepatic steatosis and fibrosis, and there were no significant differences compared to normal diet-fed hamsters. There were significant augmentations in angiotensin II and malondialdehyde, and gene expressions of collagen I, collagen III, α-SMA and Rac-1 in the placebo-treated hamsters at 24 weeks; however, these levels were reduced to normal levels in the TY-51469-treated hamsters. TY-51469 not only prevented the progression of hepatic steatosis and fibrosis, but also ameliorated hepatic steatosis and fibrosis.

Effects of compound 861 on gene expression of interstitial collagenase and type I collagen in HSC-T6 cell

Objective To investigate the effects of compound 861 on gene expression of interstitial collagenase and type I collagen in HSC-T6 cell. Methods Hepatic stellate cell （ HSC-T6 ） was cultured in medium containing different concentration of compound 861 （1.00, 0. 50, 0. 25 g/L） and cells were collected after incubation for 48 hours. Total RNA of HSC was isolated and gene expression levels of interstitial collagenase （ MMP13 ） and type I collagen were measured by reverse transcription polymerase chain reaction （RT-PCR）. Results MMP13 gene expression level of different groups of compound 861 （ 1.00, 0. 50, 0. 25 g/L） were 1.03 ±0. 16, 0. 60 ±0. 08, 0. 33 ±0.04 respectively and that of control group was 0. 26 ±0. 04. Type I collagen gene expression level of different groups of compound 861 （ 1.00, 0. 50, 0. 25 g/L） were 0. 12 ±0. 01, 0. 34 ± 0. 04, 0. 67 ± 0. 10 respectively and that of control group was 3.12±0. 46. Conclusion Compound 861 may significantly enhance MMP13 gene expression of HSC-T6, but inhibit type I collagen gene expression remarkably and these effects were correlate with the dosage of compound 861. Key words: Compound 861; HSC-T6 cell; Interstitial collagenase; Type I collagen; Gene expression

Regulation of salvia miltiorrhiza on gene expression of interstitial collagenase and type I collagen in HSC-T6-cell

Objective To investigate the effects of salvia miltiorrhiza on gene expression of interstitial collagenase and type I collagen in HSC-T6 cell. Methods Hepatic stellate cell （ HSC-T6 ） was cultured in medium containing different concentration of salvia mihiorrhiza （0. 8, 0. 4 g/L） and cells were collected after＂ incubation for 48 hours. Total RNA of HSC was isolated and gene expression levels of interstitial collagenase （MMP13） and type I c？llagen were measured by reverse transcription polymerase chain reaction. Results MMP13 gene expression level of different groups of salvia mihiorrhiza （0. 8, 0. 4 g/L） were 0. 25 ＋ 0. 05, 0. 27 ± 0. 06 respectively and that of control group was 0. 26 ± 0. 04. Type I collagen gene expression level of different groups of salvia mihiorrhiza （0. 8, 0. 4 g/L） were 0. 76 ＋0. 10, 1.55± 0.23 respectively and that of control group was 3.12 ±0.46. Conclusion Salvia miltiorrhiza may significantly inhibit type I collagen gene expression of HSC-T6 and the effects were correlate with the dosage. Sal- via mihiorrhiza has no effect on MMP13 gene expression. Key words: Salvia mihiorrhiza; HSC-T6 cell; Interstitial collagenase ; Type I collagen ; Gene expression

OR05-4 Positive association of serum prolactin concentrations with all-cause and cardiovascular mortality: Results from a 10-year follow-up of the Study of Health Pomerania (SHIP)

of ECM deposition in bGH mice. ECM deposition in adipose tissue was assessed in WAT collected from bGH versus littermate control mice at 10, 26, and 62 weeks of age, in both males and females. Adipose tissue samples from subcutaneous, perigonadal, retroperitoneal, and mesenteric depots were fixed and embedded in paraffin blocks and stained with pico-sirius red. When assessing overall collagen content, the bGH mice showed dramatic increases in ECM deposition when compared to the WT controls at most ages and in both sexes. When comparing different depots, collagen staining appeared most prevalent in the subcutaneous depot as compared to intra-abdominal depots in both male and female mice. Based on these results, analysis of collagen gene expression levels will be conducted. Specifically, collagens I, III, IV, V and VI in the subcutaneous, epididymal, mesenteric, and retroperitoneal adipose tissue depots of 6 month-old male mice will be examined through quantitative PCR analysis and results will be reported. Our histological results demonstrate a novel role of GH in adipose tissue function and suggest that ECM deposition likely contributes to the WAT depot specific differences previously reported in mice with modified GH action. Additional studies will be conducted in other GH transgenic mouse models to further establish the connection between GH and the collagen content of WAT and to deepen our understanding of the role of the ECM in WAT dysfunction.

UVA irradiation following treatment with topical 8-methoxypsoralen improves bleomycin-induced scleroderma in a mouse model, by reducing the collagen content and collagen gene expression levels in the skin

Recent studies have demonstrated that systemic or topical PUVA therapy, i.e., ultraviolet A (UVA) irradiation following treatment with 8-methoxypsoralen (8-MOP), is effective against the sclerotic skin lesions in systemic sclerosis. However, the mechanisms still remain unknown. To clarify the mechanisms of this therapy, we created a mouse model of bleomycin (BLM) injection-induced scleroderma and evaluated the effects of PUVA on the fibrotic lesions of scleroderma in this mouse model. BLM was injected subcutaneously once a day into the mice for 24 days. During the injection period, one group of mice was irradiated with UVA following local application of 8-MOP. Control groups were also set up, which were injected with phosphate-buffered saline, instead of BLM. Skin tissue samples examined histopathologically changes, measured of the content of hydroxyproline, and checked for the expression of genes encoding type I collagen, type III collagen, and transforming growth factor-β1 (TGF-β1). The mouse models of scleroderma was found to show an increase in the density of the collagen fibers and thickening of the dermis and increased expressions of type I collagen, type III collagen, and TGF-β1. However, the combination of BLM treatment and topical PUVA treatment mice appeared reduced the dermal thickness and hydroxyproline content, down-regulation of expressions of the type I and type III collagen genes was observed while the expression of the TGF-β1 gene remained unchanged. These results suggest that the effectiveness of topical PUVA therapy is attributable to the down-regulation of the expressions of the collagen genes by this treatment. The results additionally suggest that is not mediated by down-regulated expression of the TGF-β1.