Protective effect of intervention with cannabinoid type-2 receptor agonist JWH133 on pulmonary fibrosis in mice / 中华内科杂志

Objective: JWH133, a cannabinoid type 2 receptor agonist, was tested for its ability to protect mice from bleomycin-induced pulmonary fibrosis. Methods: By using a random number generator, 24 C57BL/6J male mice were randomly divided into the control group, model group, JWH133 intervention group, and JWH133+a cannabinoid type-2 receptor antagonist (AM630) inhibitor group, with 6 mice in each group. A mouse pulmonary fibrosis model was established by tracheal instillation of bleomycin (5 mg/kg). Starting from the first day after modeling, the control group mice were intraperitoneally injected with 0.1 ml of 0.9% sodium chloride solution, and the model group mice were intraperitoneally injected with 0.1 ml of 0.9% sodium chloride solution. The JWH133 intervention group mice were intraperitoneally injected with 0.1 ml of JWH133 (2.5 mg/kg, dissolved in physiological saline), and the JWH133+AM630 antagonistic group mice were intraperitoneally injected with 0.1 ml of JWH133 (2.5 mg/kg) and AM630 (2.5 mg/kg). After 28 days, all mice were killed; the lung tissue was obtained, pathological changes were observed, and alveolar inflammation scores and Ashcroft scores were calculated. The content of type Ⅰ collagen in the lung tissue of the four groups of mice was measured using immunohistochemistry. The levels of interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) in the serum of the four groups of mice were measured using enzyme-linked immunosorbent assay (ELISA), and the content of hydroxyproline (HYP) in the lung tissue of the four groups of mice was measured. Western blotting was used to measure the protein expression levels of type Ⅲ collagen, α-smooth muscle actin (α-SMA), extracellular signal regulated kinase (ERK1/2), phosphorylated P-ERK1/2 (P-ERK1/2), and phosphorylated ribosome S6 kinase type 1 (P-p90RSK) in the lung tissue of mice in the four groups. Real-time quantitative polymerase chain reaction was used to measure the expression levels of collagen Ⅰ, collagen Ⅲ, and α-SMA mRNA in the lung tissue of the four groups of mice. Results: Compared with the control group, the pathological changes in the lung tissue of the model group mice worsened, with an increase in alveolar inflammation score (3.833±0.408 vs. 0.833±0.408, P<0.05), an increase in Ashcroft score (7.333±0.516 vs. 2.000±0.633, P<0.05), an increase in type Ⅰ collagen absorbance value (0.065±0.008 vs. 0.018±0.006, P<0.05), an increase in inflammatory cell infiltration, and an increase in hydroxyproline levels [(1.551±0.051) μg/mg vs. (0.974±0.060) μg/mg, P<0.05]. Compared with the model group, the JWH133 intervention group showed reduced pathological changes in lung tissue, decreased alveolar inflammation score (1.833±0.408, P<0.05), decreased Ashcroft score (4.167±0.753, P<0.05), decreased type Ⅰ collagen absorbance value (0.032±0.004, P<0.05), reduced inflammatory cell infiltration, and decreased hydroxyproline levels [(1.148±0.055) μg/mg, P<0.05]. Compared with the JWH133 intervention group, the JWH133+AM630 antagonistic group showed more severe pathological changes in the lung tissue of mice, increased alveolar inflammation score and Ashcroft score, increased type Ⅰ collagen absorbance value, increased inflammatory cell infiltration, and increased hydroxyproline levels. Compared with the control group, the expression of α-SMA, type Ⅲ collagen, P-ERK1/2, and P-p90RSK proteins in the lung tissue of the model group mice increased, while the expression of type Ⅰ collagen, type Ⅲ collagen, and α-SMA mRNA increased. Compared with the model group, the protein expression of α-SMA (relative expression 0.60±0.17 vs. 1.34±0.19, P<0.05), type Ⅲ collagen (relative expression 0.52±0.09 vs. 1.35±0.14, P<0.05), P-ERK1/2 (relative expression 0.32±0.11 vs. 1.14±0.14, P<0.05), and P-p90RSK (relative expression 0.43±0.14 vs. 1.15±0.07, P<0.05) decreased in the JWH133 intervention group. The type Ⅰ collagen mRNA (2.190±0.362 vs. 5.078±0.792, P<0.05), type Ⅲ collagen mRNA (1.750±0.290 vs. 4.935±0.456, P<0.05), and α-SMA mRNA (1.588±0.060 vs. 5.192±0.506, P<0.05) decreased. Compared with the JWH133 intervention group, the JWH133+AM630 antagonistic group increased the expression of α-SMA, type Ⅲ collagen, P-ERK1/2, and P-p90RSK protein in the lung tissue of mice, and increased the expression of type Ⅲ collagen and α-SMA mRNA. Conclusion: In mice with bleomycin-induced pulmonary fibrosis, the cannabinoid type-2 receptor agonist JWH133 inhibited inflammation and improved extracellular matrix deposition, which alleviated lung fibrosis. The underlying mechanism of action may be related to the activation of the ERK1/2-RSK1 signaling pathway.

Establishment of a reporter system for estimating activation of human hepatic stellate cells based on COL1A1 promoter and enhanced green fluorescent protein / 北京大学学报(医学版)

OBJECTIVE@#To establish a visual reporting system for evaluating the activity of collagen Ⅰ α 1 chain (COL1A1) gene promoter in immortalized human hepatic stellate cells, so as to estimate the activation status of the cells and provide a new cell model for the screening and study of anti-hepatic fibrosis drugs.@*METHODS@#The promoter sequence of human COL1A1 was amplified from the genomic DNA of human hepatocarcinoma cell line HepG2. Based on the pLVX-AcGFP1-N1 plasmid, the recombinant plasmid pLVX-COL1A1-enhanced green fluorescent protein (EGFP) was constructed, in which the enhanced green fluorescent protein gene expression was regulated by the COL1A1 promoter. The monoclonal cell line was acquired by stably transfecting pLVX-COL1A1-EGFP into the immortalized human hepatic stellate cell line LX-2 by the lentivirus packaging system and screening. The cell line was treated with transforming growth factor-β1 (TGF-β1) or co-treated with TGF-β1 and drugs with potential anti-hepatic fibrosis effects. The EGFP fluorescence intensity in cells was analyzed by the fluorescence microscope and ImageJ 1.49 software using a semi-quantitative method. The COL1A1 and EGFP mRNA were detected by reverse transcription real-time quantitative PCR (RT-qPCR), and corresponding proteins were detected by Western blot.@*RESULTS@#The recombinant plasmid pLVX-COL1A1-EGFP with the expression of EGFP regulated by COL1A1 promoter was successfully constructed. Kozak sequence was added to enhance the expression of EGFP, which was identified by double digestion and sequencing. The LX-2 monoclonal cell line LX-2-CE stably transfected with pLVX-COL1A1-EGFP was obtained. After co-treatment with TGF-β1 and 5 μmol/L dihydrotanshinone Ⅰ with potential anti-hepatic fibrosis effect for 24 h, the total fluorescence intensity and the average fluorescence intensity of LX-2-CE were lower than those in TGF-β1 single treatment group (P < 0.05), the intracellular mRNA and protein levels of COL1A1 and EGFP were also lower than those in the TGF-β1 single treatment group (P < 0.05).@*CONCLUSION@#A reporter system for estimating activation of hepatic stellate cells based on COL1A1 promoter regulated EGFP expression is successfully constructed, which could visually report the changes in COL1A1 expression, one of the activation-related markers of hepatic stellate cells, in vitro. It provides a new cell model for the screening and study of anti-hepatic fibrosis drugs.

Low-dose paclitaxel modulates the cross talk between the JNK and Smad signaling in primary biliary fibroblasts

SUMMARY OBJECTIVE: The objective of this study was to explore the molecular mechanism underlying the occurrence of benign bile duct stricture and the target of low-dose paclitaxel in the prevention of benign bile duct stricture. METHODS: Under the stimulation of transforming growth factor beta 1, the expression of collagen type I and connective tissue growth factor were detected on isolated primary fibroblasts. The phosphorylation levels of JNK and Smad2L were detected using Western blot. The effect of low-dose paclitaxel on the transforming growth factor beta 1-induced inhibition of type I collagen and connective tissue growth factor expression and JNK and Smad2L phosphorylation was also observed. RESULTS: Transforming growth factor beta 1 induced the secretion of type I collagen and connective tissue growth factor as well as JNK phosphorylation in biliary fibroblasts. The JNK inhibitor or siRNA-Smad2 inhibited the transforming growth factor beta 1-induced secretion of type I collagen and connective tissue growth factor. Low-dose paclitaxel inhibited the expression of type I collagen induced by transforming growth factor beta 1 and may inhibit the secretion of collagen in biliary fibroblasts. CONCLUSION: The activation of JNK/Smad2L induced by transforming growth factor beta 1 is involved in the occurrence of benign bile duct stricture that is mediated by the overexpression of type I collagen and connective tissue growth factor, and low-dose paclitaxel may inhibit the phosphorylation of JNK/Smad2L.

Cranial bone regeneration with collagen type I and polyvinylpyrrolidone (Fibroquel®) combined with hyaluronic acid in Wistar rats: morphological study / Regeneración ósea craneal con colágeno tipo I y polivinilpirrolidona (Fibroquel®) combinado con ácido hialurónico en ratas Wistar: estudio morfológico

SUMMARY: The purpose of this study was to evaluate by morphological methods, if a mixture of Fibroquel® and hyaluronic acid implanted in an animal model of cranial bone injury could promote bone regeneration. 12 Wistar rats were divided in three groups, control group, bone injury without treatment and bone injury with treatment. After experimental period, bone samples were taken and stained with H & E, Masson trichrome, PAS-D, immunohistochemistry with anti-PCNA monoclonal antibody and applied a semiquantitative morphometric method. Treatment group showed extensive areas of collagen fibers in contact with normal bone tissue, areas of normal histology, PAS positive material and less cellular proliferation. We demonstrated for the first time that a mixture of Fibroquel® and hyaluronic acid implanted in an animal model of cranial bone injury promotes bone regeneration.

RESUMEN: El propósito de este estudio fue evaluar por métodos morfológicos, si una mezcla de Fibroquel® y ácido hialurónico implantado en un modelo animal de lesión del hueso craneal podría promover la regeneración ósea. Se dividieron 12 ratas Wistar en tres grupos, grupo control, lesión ósea sin tratamiento y lesión ósea con tratamiento. Después del período experimental, se tomaron muestras de hueso y se tiñeron con H & E, tricrómico de Masson, PAS-D, inmunohistoquímica con anticuerpo monoclonal anti-PCNA y se aplicó un método morfométrico semicuantitativo. El grupo de tratamiento mostró áreas extensas de fibras de colágeno en contacto con tejido óseo normal, áreas de histología normal, material PAS positivo y menor proliferación celular. Demostramos por primera vez que una mezcla de Fibroquel® y ácido hialurónico implantado en un modelo animal de lesión del hueso craneal promueve la regeneración ósea.

Effects of the application of the amniotic membrane in the healing process of skin wounds in rats

Abstract Purpose: To evaluate the efficacy of the application of the human amniotic membrane (HAM) on the inflammatory process, fibroblast proliferation, formation of collagenand reduction of skin wound areas in rats. Methods: Thirty six rats were submitted to a surgical injury induction and divided into two groups (n = 18): group C (control) and T (treated with the HAM). The macroscopic evolution in the wound area and the histological characteristics of the skin samples were evaluated. Results: The regression of the wound area was greater in group T. The histological analysis revealed a significant reduction (p < 0.05) in the inflammatory infiltrate in group T at all experimental periods compared with that in the control group. Furthermore, the group T presented a significant increase in the proliferation of fibroblasts at 14 and 21 days compared with group C (p < 0.05). Regarding the deposition of mature collagen fibers, there was an increase in the replacement of type III collagen by type I collagen in group T (p < 0.05). Conclusion: Treatment with the HAM reduced the healing time as well as the inflammatory responses, increased the proliferation of fibroblasts, and induced a higher concentration of mature collagen fibers.

Human plasma fibronectin promotes proliferation and differentiation of odontoblast

Abstract Objective To assess the effect of fibronectin (Fn) and porcine type I collagen (PCOL) on odontoblast-like cells in vitro. Material and Methods Rat odontoblast-like cells (MDPC-23 cells) were inoculated and cultured on Fn-coated or type I collagen-coated substrates. Proliferation assay, alkaline phosphatase activity (ALP activity), mRNA expression of hard tissue-forming markers, and Alizarin red staining were investigated over a period of 10 days. Results Cells maintained a high proliferation activity on Fn and PCOL even at a low seeding concentration (0.5×104/mL) as demonstrated by CCK-8 assay. The proliferation activity of cells on Fn increases in a concentration-dependent manner while it reached a plateau after 10 µg/mL. Cells adopted long, thin and spindle shape on Fn(10-50) and PCOL. Parallel actin filaments were observed in MDPC-23 cells cultured on Fn and PCOL. ALP activity was markedly up-regulated on Fn and PCOL-coated surfaces. Importantly, gene expression of BSP (Fn10: 2.44±0.32; Fn20: 3.05±0.01; Fn30: 2.90±0.21; Fn40: 2.74±0.30; Fn50: 2.64±0.12; PCOL: 2.20±0.03) and OCN (Fn10: 2.52±0.23; Fn20: 2.28±0.24; Fn30: 2.34±0.21; Fn40: 2.34±0.25; Fn50: 2.20±0.22; PCOL: 1.56±0.16) was significantly enhanced on Fn and PCOL substrates as compared with control; moreover, expression of integrin beta 1 (ITGB1), an ubiquitous cell surface receptor was augmented in Fn(10-50) and PCOL groups simultaneously. In accordance with the ALP activity and gene expression data, calcific deposition in cells grown on Fn(10-50) and PCOL was observed as well. Conclusion Despite the limitation of this study, the findings indicate that a surface coating of Fn enhances the proliferation, differentiation and mineralization of odontoblast-like cells by activation of integrin beta 1 (ITG B1). The promoting effects of Fn on MDPC-23 cells were achieved at a comparatively lower coating concentration than type I collagen (300 µg/mL). Specifically, it is suggested that the optimum coating concentration of Fn to be 10 µg/mL.

Non-cross-linked collagen type I/III materials enhance cell proliferation: in vitro and in vivo evidence

Objective: To analyze Mucograft®(MG), a recently introduced collagen matrix, in vitro and in vivo, and compare it with BioGide®(BG), a well-established collagen membrane, as control. Material and Methods: A detailed analysis of the materials surface and ultra-structure was performed. Cellular growth patterns and proliferation rates of human fibroblasts on MG and BG were analyzed in vitro. In addition, the early tissue reaction of CD-1 mouse to these materials was analyzed by means of histological and histomorphometrical analysis. Results: MG showed a three-fold higher thickness both in dry and wet conditions, when compared to BG. The spongy surface of BG significantly differed from that of MG. Cells showed a characteristic proliferation pattern on the different materials in vitro. Fibroblasts tended to proliferate on the compact layers of both collagens, with the highest values on the compact side of BG. In vivo, at day three both materials demonstrated good tissue integration, with a mononuclear cell sheet of fibroblasts on all surfaces, however, without penetrating into the materials. Conclusions: The findings of this study showed that MG and BG facilitate cell proliferation on both of their surfaces in vitro. In vivo, these two materials induce a comparable early tissue reaction, while serving as cell occlusive barriers. .

Mycobacterial laminin-binding histone-like protein mediates collagen-dependent cytoadherence

When grown in the presence of exogenous collagen I, Mycobacterium bovis BCG was shown to form clumps. Scanning electron microscopy examination of these clumps revealed the presence of collagen fibres cross-linking the bacilli. Since collagen is a major constituent of the eukaryotic extracellular matrices, we assayed BCG cytoadherence in the presence of exogenous collagen I. Collagen increased the interaction of the bacilli with A549 type II pneumocytes or U937 macrophages, suggesting that BCG is able to recruit collagen to facilitate its attachment to host cells. Using an affinity chromatography approach, we have isolated a BCG collagen-binding protein corresponding to the previously described mycobacterial laminin-binding histone-like protein (LBP/Hlp), a highly conserved protein associated with the mycobacterial cell wall. Moreover, Mycobacterium leprae LBP/Hlp, a well-characterized adhesin, was also able to bind collagen I. Finally, using recombinant fragments of M. leprae LBP/Hlp, we mapped the collagen-binding activity within the C-terminal domain of the adhesin. Since this protein was already shown to be involved in the recognition of laminin and heparan sulphate-containing proteoglycans, the present observations reinforce the adhesive activities of LBP/Hlp, which can be therefore considered as a multifaceted mycobacterial adhesin, playing an important role in both leprosy and tuberculosis pathogenesis.

In vitro action of leukemia inhibitory factor (LIF) on mid-secretory stage endometrial stromal cells collected from hormone-simulated, ovariectomized monkey and maintained in three-dimensional primary culture.

Leukemia inhibitory factor (LIF) is a pleiotropic cytokine that is known to play an important role in blastocyst implantation. The putative action of LIF in the regulation of uterine function has been examined using mid-secretory stage monkey endometrial stromal cells cultured on rat-tail collagen type I and treated with recombinant human LIF (rhLIF) or immunoneutralized LIF (in LIF) under serum-free condition. Long-term ovariectomized rhesus monkeys (n=8) underwent simulation of their menstrual cycles with steroid hormones and endometrial tissue samples were collected on cycle day 18; stromal cells were isolated and grown in primary culture on three-dimensional collagen matrix. Significant decline in cellular protein synthesis (P < 0.01) and cell proliferation index (P < 0.05) was observed in cells with increasing doses (0-1000 ng/ml) of rhLIF under serum-free in vitro condition. JAK1 expression in cultured cells increased (P < 0.01) in response to rhLIF as revealed from Western blot and confocal laser scanning microscopic examination, STAT1 and STAT2 expressions were unchanged, while pSTAT3 expression increased (P < 0.01) with increased concentration of rhLIF in culture medium. Autophosphorylation of JAK1 in endometrial stromal cells showed no change with increasing concentration (0.01 to 100 ng/ml) of rhLIF in vitro, but significant (P < 0.05) increase was observed with the time of exposure to rhLIF. Immunoneutralization of LIF or no addition of rhLIF to cultured cells led to significant (P < 0.01) increase in stromal cell proliferation index and significant (P < 0.01) decrease in the level of JAK1 and its autophosphorylation as compared to cells exposed to rhLIF alone. From the present set of experiments we conclude that rhLIF affects the physiological behaviour of monkey mid secretory stage endometrial stromal cells in vitro via the JAK-STAT signaling pathway.