Connective Tissue Growth Factor Expression Research Articles

The regulatory effect of electroacupuncture on endometrial M1-type macrophages in rats with intrauterine adhesions.

To observe the effect of electroacupuncture(EA) on endometrial fibrosis and M1-type macrophages in rats with intrauterine adhesions(IUA), so as to explore the possible mechanism of EA in the treatment of IUA. Fifteen female SD rats were randomly divided into blank group, model group and EA group, with 5 rats in each group. The IUA rat model was established by double damage method using mechanical scraping combined with lipopolysaccharide infection. Rats in the EA group were treated with acupuncture at "Guanyuan"(CV4), and EA at bilateral "Zusanli"(ST36) and "Sanyinjiao"(SP6)for 20 minutes each time, once a day, for 3 consecutive cycles of estrus. Five rats in each group were sampled during the estrous period, and the endometrial morphology, endometrial thickness and the number of blood vessels and glands were observed after HE staining. The fibrotic area of the uterus was observed after Masson staining. The positive expressions of Runt-related transcription factor(RUNX1), transforming growth factor-β1(TGF-β1), connective tissue growth factor(CTGF), α-smooth muscle actin(α-SMA), collagen type I(Col-Ⅰ), cluster of differentiation 86(CD86), interleukin-1β(IL-1β), and tumor necrosis factor-α(TNF-α) in endometrial tissue were detected by immunohistochemistry. Western blot was used to detect relative protein expressions of RUNX1, TGF-β1, α-SMA, CD86, and TNF receptor 2 (TNFR2), and real-time fluorescence quantitative PCR was used to detect mRNA expressions of RUNX1, TGF-β1, α-SMA, CD86, and TNF-α in the endometrium. During the estrous phase, the endometrial layer in the model group was damaged, with reduced folds, disordered arrangement of epithelial cells, loose fibrous connective tissue, significant narrowing and adhesions in the uterine cavity, interstitial congestion, edema, and a significant infiltration of inflammatory cells with sparse glands. While uterine tissue structure of the EA group was basically intact, resembling a normal uterus, with more newly formed glands and a small amount of inflammatory cell infiltration. In comparison with the blank group, the endometrial thickness, the number of blood vessels, and the number of glands were significantly decreased(P<0.001) in the model group, while the ratio of uterine fibrosis area, the positive expressions of RUNX1, TGF-β1, CTGF, α-SMA, Col-Ⅰ, CD86, IL-1β, and TNF-α, the protein relative expressions of RUNX1, TGF-β1, α-SMA, CD86 and TNFR2, and the mRNA relative expression levels of RUNX1, TGF-β1, α-SMA, CD86 and TNF-α in the endometrium were significantly increased (P<0.001, P<0.01). Compared to the model group, the endometrial thickness, the number of blood vessels, and the number of glands were significantly increased(P<0.01, P<0.05) in the EA group, while the ratio of uterine fibrosis area, the positive expressions of RUNX1, TGF-β1, CTGF, α-SMA, Col-Ⅰ, CD86, IL-1β and TNF-α in the endometrial tissue, the protein expressions of RUNX1, TGF-β1, α-SMA, CD86 and TNFR2, and the mRNA relative expressions of RUNX1, TGF-β1, α-SMA, CD86 and TNF-α in the endometrium were significantly decreased (P<0.001, P<0.01, P<0.05). EA can improve endometrial fibrosis in IUA rats, which may be related to its function in decreasing the level of endometrial M1-type macrophages and the secretion of related inflammatory factors.

Blocking the MIR155HG/miR-155 axis reduces CTGF-induced inflammatory cytokine production and α-SMA expression via upregulating AZGP1 in hypertrophic scar fibroblasts

Hypertrophic scarring (HS) is a pathological condition characterized by excessive fibrosis and inflammation, resulting in excessive extracellular matrix formation in the skin. MIR155HG, a long non-coding RNA, is abnormally upregulated in fibrotic tissues; however, its underlying mechanism is poorly understood. Using single-cell sequencing data, we analyzed connective tissue growth factor (CTGF) expression in various cell types in HS and normal skin tissues and MIR155HG expression in clinical samples. To investigate the mechanism of fibrosis, an in vitro model using CTGF-treated hypertrophic scar fibroblasts (HSFBs) was established and qRT-PCR, western blotting and ELISA assays were performed to investigate the expression of interleukin (IL)-1β, IL-6, and mesenchymal markers α-smooth muscle actin (α-SMA). CTGF stimulates MIR155HG level through phosphorylated STAT3 binding to the MIR155HG promoter. We analyzed the methylation of MIR155HG, assessed the levels of miR-155-5p/−3p in CTGF-treated HSFBs and identified differentially expressed genes among HS and NS samples using the Gene Expression Omnibus RNA sequencing data. The binding between miR-155-5p/−3p and AZGP1 was confirmed using a dual-luciferase assay and inflammatory cytokine production and α-SMA expression were investigated in rescue experiments. The findings revealed that CTGF elevated inflammatory cytokine production, α-SMA and MIR155HG expression in HSFBs. MIR155HG is upregulated in HS tissues due to low DNA methylation. Mechanistically, miR-155-5p/−3p was directly bound to MIR155HG 3’UTR. MIR155HG silencing inhibited cytokine production and α-SMA expression by repressing the generation of miR-155-5p/−3p in CTGF-treated HSFBs. Bioinformatics analysis and luciferase reporter assays revealed that miR-155-5p/−3p targets AZGP1. In addition, transfection with plasmids carrying AZGP1 cDNA significantly inhibited the signaling activity of miR-155-5p/−3 p-overexpressing HSFBs. Our findings highlight the importance of the MIR155HG/miR-155/AZGP1 axis in regulating cytokine production and α-SMA in HS.

Neutralization of CX3CL1 Attenuates TGF-β-Induced Fibroblast Differentiation Through NF-κB Activation and Mitochondrial Dysfunction in Airway Fibrosis.

Severe asthma, characterized by inflammation and airway remodeling, involves fibroblast differentiation into myofibroblasts expressing α-SMA. This process leads to the production of fibronectin and connective tissue growth factor (CTGF), driven by factors such as transforming growth factor (TGF)-β. Furthermore, the persistent presence of myofibroblasts is associated with resistance to apoptosis and mitochondrial dysfunction. The chemokine (C-X3-C motif) ligand 1 (CX3CL1) plays a role in tissue fibrosis. However, it is currently unknown whether neutralization of CX3CL1 decreases TGF-β-induced fibroblast differentiation and mitochondrial dysfunction in normal human lung fibroblasts (NHLFs). CX3CL1/C-X3-C motif chemokine receptor 1 (CX3CR1), CX3CL1 was analyzed by immunofluorescence (IF) or immunohistochemical (IHC) staining of ovalbumin-challenged mice. CX3CL1 release was detected by ELISA. TGF-β-induced CTGF, fibronectin, and α-SMA expression were evaluated in NHLFs following neutralization of CX3CL1 (TP213) treatment for the indicated times by Western blotting or IF staining. Mitochondrion function was detected by a JC-1 assay and seahorse assay. Cell apoptosis was observed by a terminal uridine nick-end labeling (TUNEL) assay. An increase in CX3CL1 expression was observed in lung tissues from mice with ovalbumin-induced asthma by IF staining. CX3CR1 was increased in the subepithelial layer of the airway by IHC staining. Moreover, CX3CR1 small interfering (si)RNA downregulated TGF-β-induced CTGF and fibronectin expression in NHLFs. CX3CL1 induced CTGF and fibronectin expression in NHLFs. TGF-β-induced CX3CL1 secretion from NHLFs. Furthermore, TP213 decreased TGF-β-induced CTGF, fibronectin, and α-SMA expression in NHLFs. Mitochondrion-related differentially expressed genes (DEGs) were examined after CX3CL1 neutralization in TGF-β-treated NHLFs. TP213 alleviated TGF-β-induced mitochondrial dysfunction and apoptosis resistance in NHLFs. CX3CL1 induced p65, IκBα, and IKKα phosphorylation in a time-dependent manner. Furthermore, CX3CL1-induced fibronectin expression and JC-1 monomer were decreased by p65 siRNA. TP213 reduced TGF-β-induced p65 and α-SMA expression in NHLFs. These findings suggest that neutralizing CX3CL1 attenuates lung fibroblast activation and mitochondrial dysfunction. Understanding the impacts of CX3CL1 neutralization on fibroblast mitochondrial function could contribute to the development of therapeutic strategies for managing airway remodeling in severe asthma.

Prognostic significance of connective tissue growth factor expression in stromal cells in patients with diffuse‑type gastric cancer.

Connective tissue growth factor (CTGF) is a target gene of the Hippo signaling pathway. Its differential role in the histological types of gastric cancer (GC) remains unknown; therefore, the present study aimed to confirm the clinical significance of CTGF expression in cancer and stromal cells in patients with GC depending on the histological type. The present study enrolled 589 patients with GC. Immunohistochemistry was used to analyze CTGF expression in cancer and stromal cells. CTGF mRNA expression data and the corresponding clinical information of GC samples were collected from The Cancer Genome Atlas (TCGA) database. Subsequently, the associations between CTGF expression and several clinicopathological factors were investigated. In the present study, CTGF expression was mainly observed in the cytoplasm of cancer and stromal cells. CTGF expression in stromal cells was significantly associated with CTGF expression in cancer cells (P<0.001). CTGF positivity in stromal cells was also significantly associated with intestinal type, non-scirrhous type, tumor depth (T1-2), lymph node metastasis (negative), lymphatic invasion (negative) and tumor size (<5 cm). Low CTGF expression in stromal cells was independently associated with worse overall survival (OS). Furthermore, the OS of patients with low CTGF expression in stromal cells, especially in patients with diffuse-type GC, was significantly worse than patients with high CTGF expression (P=0.022). This trend was similar to that revealed by TCGA data analysis. In conclusion, low CTGF expression was associated with a significantly worse OS in patients with diffuse-type GC. These data indicated that CTGF, and its control by the Hippo pathway, may be considered potential treatment targets in diffuse-type GC.

Inhibition of Connective Tissue Growth Factor Expression in Adult Retinal Pigment Epithelial-19 Cells by Blocking Yes-Associated Protein/Transcriptional Coactivator with PDZ-Binding Motif Activity.

Purpose: To investigate the effect of yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) on connective tissue growth factor (CTGF) expression in adult retinal pigment epithelial (ARPE)-19 cells. We also studied the inhibitory effect of K-975, a new pan-transcriptional enhanced associate domain (TEAD) inhibitor, and luteolin, a plant-derived flavonoid on CTGF expression. Methods: ARPE-19 cells were transfected with either YAP or TAZ overexpression plasmid or treated with transforming growth factor (TGF)-β2. The cells were cultured either with or without K-975 or luteolin. The expression of YAP, TAZ, and CTGF was examined using real-time PCR. Results: ARPE-19 cells overexpressing YAP or TAZ exhibited significantly increased CTGF expression. This increase was attenuated by K-975 or luteolin alone. TGF-β2 treatment significantly raised the expression of not just YAP and TAZ, but also CTGF in ARPE-19 cells. TGF-β2 treatment-enhanced CTGF expression was considerably lowered by the addition of K-975 or luteolin. Conclusions: Overexpression of YAP or TAZ and treatment with TGF-β2 led to an increase in the expression of CTGF in ARPE-19 cells. These increases were attenuated by treatment with K-975 and luteolin. These findings suggest that YAP and TAZ may be related to the expression of CTGF in ARPE-19 cells and that K-975 and luteolin can be explored as potential therapeutic agents for preventing CTGF production in vitreoretinal fibrosis.

Impact of umbelliprenin-containing niosome nanoparticles on VEGF-A and CTGF genes expression in retinal pigment epithelium cells.

To investigate the impact of niosome nanoparticles carrying umbelliprenin (UMB), an anti-angiogenic and anti-inflammatory plant compound, on the expression of vascular endothelial growth factor (VEGF-A) and connective tissue growth factor (CTGF) genes in a human retinal pigment epithelium (RPE)-like retina-derived cell line. UMB-containing niosomes were created, optimized, and characterized. RPE-like cells were treated with free UMB and UMB-containing niosomes. The IC50 values of the treatments were determined using an MTT assay. Gene expression of VEGF-A and CTGF was evaluated using real-time polymerase chain reaction after RNA extraction and cDNA synthesis. Niosomes' characteristics, including drug entrapment efficiency, size, dispersion index, and zeta potential were assessed. Free UMB had an IC50 of 96.2 µg/mL, while UMB-containing niosomes had an IC50 of 25 µg/mL. Treatment with UMB-containing niosomes and free UMB resulted in a significant reduction in VEGF-A expression compared to control cells (P=0.001). Additionally, UMB-containing niosomes demonstrated a significant reduction in CTGF expression compared to control cells (P=0.05). However, there was no significant reduction in the expression of both genes in cells treated with free UMB. Both free UMB and niosome-encapsulated UMB inhibits VEGF-A and CTGF genes expression. However, the latter demonstrates significantly greater efficacy, potentially due to the lower UMB dosage and gradual delivery. These findings have implications for anti-angiogenesis therapeutic approaches targeting age-related macular degeneration.

MicroRNA-143 targets SIRT2 to mediate the histone acetylation of PLAUR and modulates functions of astrocytes in spinal cord injury

This study aimed to explore effects of microRNA (miR)-143 on the proliferation, apoptosis, and cytokine secretion in astrocytes after spinal cord injury (SCI). After gain- and loss-of-function assays and transforming growth factor (TGF)-β stimulation in astrocytes, the cell viability, proliferation, and apoptosis were examined. The expression of miR-143, SIRT2, and PLAUR and levels of astrocyte-related glial fibrillary acidic protein (GFAP), Vimentin, chondroitin sulfate proteoglycan (CSPG), and connective tissue growth factor (CTGF) were also measured. The binding relationship between miR-143 and SIRT2 was assessed, as well as the correlation of PLAUR with SIRT2. In established SCI rat models, the locomotion function and astrocyte hyperplasia were detected. The TGF-β stimulation decreased miR-143 but increased SIRT2 expression in astrocytes. Mechanistically, miR-143 negatively targeted SIRT2 and SIRT2 down-regulation inhibited the H3K27 deacetylation of PLAUR promoter to increase PLAUR expression. miR-143 up-regulation inhibited TGF-β stimulated-proliferation, promoted cell apoptosis, and reduced GFAP, Vimentin, CSPG, and CTGF expression in astrocytes, which was counterweighed by SIRT2 overexpression. SIRT2 silencing reduced the proliferation and GFAP, Vimentin, CSPG, and CTGF expression while augmenting the apoptosis in TGF-β stimulated astrocytes, which was abrogated by PLAUR silencing. The injection of miR-143 agomir improved the locomotion function and reduced the astrocyte hyperplasia in SCI rats, which was reversed by silencing PLAUR. miR-143 targeted SIRT2 to affect PLAUR expression via the regulation of histone acetylation, which repressed the astrocyte activation in vivo and in vitro to improve the locomotion function in SCI rats.

Imperatorin ameliorates pulmonary fibrosis via GDF15 expression.

Background: Pulmonary fibrosis features in damaged pulmonary structure or over-produced extracellular matrix and impaired lung function, leading to respiratory failure and eventually death. Fibrotic lungs are characterized by the secretion of pro-fibrotic factors, transformation of fibroblasts to myofibroblasts, and accumulation of matrix proteins. Hypothesis/purpose: Imperatorin shows anti-inflammatory effects on alveolar macrophages against acute lung injury. We attempt to evaluate the properties of imperatorin on the basis of fibroblasts. Methods: In in vitro, zymosan was introduced to provoke pro-fibrotic responses in NIH/3T3 or MRC-5 pulmonary fibroblasts. Imperatorin was given for examining its effects against fibrosis. The mice were stimulated by bleomycin, and imperatorin was administered to evaluate the prophylactic potential in vivo. Results: The upregulated expression of connective tissue growth factor (CTGF), α-smooth muscle actin (α-SMA), and collagen protein due to zymosan introduction was decreased by imperatorin in fibroblasts. Zymosan induced the activity of transglutaminase 2 (TGase2) and lysyl oxidase (LOX), which was also inhibited by the administration of imperatorin. Imperatorin alone enhanced sirtuin 1 (SIRT1) activity and growth differentiation factor 15 (GDF15) secretion in fibroblasts via LKB1/AMPK/CREB pathways. In addition, GDF15 exerted a beneficial effect by reducing the protein expression of CTGF, α-SMA, and collagen and the activities of TGase and LOX. Moreover, orally administered imperatorin showed prophylactic effects on bleomycin-induced pulmonary fibrosis in mice. Conclusion: Imperatorin reduces fibrotic marker expression in fibroblasts and also increases GDF15 secretion via the LKB1/AMPK/CREB pathway, attenuating pro-fibrotic responses in vitro. Imperatorin also alleviates pulmonary fibrosis induced by bleomycin in vivo.

Unraveling Connective Tissue Growth Factor as a Therapeutic Target and Assessing Kahweol as a Potential Drug Candidate in Triple-Negative Breast Cancer Treatment.

Triple-negative breast cancer (TNBC) is characterized by aggressive behavior and limited treatment options, necessitating the identification of novel therapeutic targets. In this study, we investigated the clinical significance of connective tissue growth factor (CTGF) as a prognostic marker and explored the potential therapeutic effects of kahweol, a coffee diterpene molecule, in TNBC treatment. Initially, through a survival analysis on breast cancer patients from The Cancer Genome Atlas (TCGA) database, we found that CTGF exhibited significant prognostic effects exclusively in TNBC patients. To gain mechanistic insights, we performed the functional annotation and gene set enrichment analyses, revealing the involvement of CTGF in migratory pathways relevant to TNBC treatment. Subsequently, in vitro experiments using MDA-MB 231 cells, a representative TNBC cell line, demonstrated that recombinant CTGF (rCTGF) administration enhanced cell motility, whereas CTGF knockdown using CTGF siRNA resulted in reduced motility. Notably, rCTGF restored kahweol-reduced cell motility, providing compelling evidence for the role of CTGF in mediating kahweol's effects. At the molecular level, kahweol downregulated the protein expression of CTGF as well as critical signaling molecules, such as p-ERK, p-P38, p-PI3K/AKT, and p-FAK, associated with cell motility. In summary, our findings propose CTGF as a potential prognostic marker for guiding TNBC treatment and suggest kahweol as a promising antitumor compound capable of regulating CTGF expression to suppress cell motility in TNBC. These insights hold promise for the development of targeted therapies and improved clinical outcomes for TNBC patients.

Endogenous FGF1 Deficiency Aggravates Doxorubicin-Induced Hepatotoxicity.

Doxorubicin (DOX) is a broad-spectrum antineoplastic agent that widely used in clinic. However, its application is largely limited by its toxicity in multiple organs. Fibroblast growth factor 1 (FGF1) showed protective potential in various liver diseases, but the role of endogenous FGF1 in DOX-induced liver damage is currently unknown. Both wild-type (WT) and FGF1 knockout (FGF1-KO) mice were treated with DOX. DOX induced loss of body weight and liver weight and elevation of ALT and AST in WT mice, which were aggravated by FGF1 deletion. FGF1 deletion exacerbated hepatic oxidative stress mirrored by further elevated 3-nitrosative modification of multiple proteins and malondialdehyde content. These were accompanied by blunted compensatively antioxidative responses indicated by impaired upregulation of nuclear factor erythroid 2-related factor 2 and its downstream antioxidant gene expression. The aggravated oxidative stress was coincided with exacerbated cell apoptosis in DOX-treated FGF1-KO mice reflected by further increased TUNEL positive cell staining and BCL-2-associated X expression and caspase 3 cleavage. These detrimental changes in DOX-treated FGF1-KO mice were associated with worsened intestinal fibrosis and increased upregulation fibrotic marker connective tissue growth factor and α-smooth muscle actin expression. However, DOX-induced hepatic inflammatory responses were not further affected by FGF1 deletion. These results demonstrate that endogenous FGF1 deficiency aggravates DOX-induced liver damage and FGF1 is a potential therapeutic target for treatment of DOX-associated hepatoxicity.

Cardiac-specific deletion of myosin phosphatase targeting subunit 2 alleviates cardiac fibrosis in mineralocorticoid receptor-related hypertension

Abstract Introduction Myosin phosphatase targeting subunit 2 (MYPT2) is a crucial subunit of cardiac myosin light chain (MLC) phosphatase. The phosphorylation of MLC in the ventricle plays a pivotal role in cardiac hypertrophy and function. Our previous study demonstrated that overexpression of MYPT2 activated MLC phosphatase leading to cardiac dysfunction. Mineralocorticoid receptor (MR)-related hypertension is associated with extensive cardiac fibrosis. It remains unknown whether MYPT2 may play a role in the development of cardiac fibrosis in MR-related hypertension. Purpose Our purpose is to investigate the effect of MYPT2 on cardiac fibrosis in MR-related hypertension. Methods After the knockdown of MYPT2 using MYPT2-siRNA, HL-1 cells (murine cardiomyocytes) were incubated with aldosterone (ALDO) for 24h. Cardiac-­specific MYPT2 knockout (c-MYPT2-/-) mice were generated using the Cre-lox system (αMHC-­Cre; MYPT2f/f). MR-related hypertension mice were induced in c-MYPT2-/- and wild-type (MYPT2+/+) mice with the subcutanouse infusion of ALDO and 8% NaCl food for 4 weeks after uninephrectomy. Systolic blood pressure (SBP) was measured by tail cuffs every week. WB, qPCR, cardiac echo, and histological examinations were performed. Results In HL-1 cells, ALDO increased the expression of connective tissue growth factor (CTGF), MYPT2, and the catalytic subunit of the type-1 phosphatase δ isoform (PP1cδ) in a concentration-dependent manner. MYPT2 protein levels significantly decreased by 80% in HL-1 cells transfected with MYPT2 siRNA compared with cells transfected with unspecific siRNA. Knockdown of MYPT2 significantly decreased the protein level of CTGF. In the mice model, the MYPT2 expression in the heart from MYPT2-/- mice was decreased to less than 30% compared with MYPT2+/+ mice. Heart rate, blood pressure, and cardiac systolic function were normal in c-MYPT2-/- mice. PP1cδ was decreased in c-MYPT2-/- mice compared with MYPT2+/+ mice. The expression of cardiac MLC kinase was not changed. The level of phosphorylation of MLC in c-MYPT2-/- mice was significantly higher than MYPT2+/+. Blood pressure elevation and left ventricular hypertrophy were observed in both MR-related hypertension mice. No significant difference in heart size and the nuclear localization of MR in cardiomyocytes. However, ejection fraction and fractional shortening in echocardiography were higher in c-MYPT2-/- than in MYPT2+/+ in MR-related hypertension mice. Histopathological examinations revealed the degree of fibrosis in c-MYPT2-/- mice was lower than in MYPT2+/+ mice with the decreased expression of CTGF. Conclusions Cardiac-specific deletion of MYPT2 resulted in a decrease of MLC phosphatase and an increase of phosphorylation in MLC in vivo. MYPT2 deletion can inhibit cardiac fibrosis in a MR-related hypertension model.

P2 X 7 receptor is a critical regulator of extracellular ATP-induced profibrotic genes expression in rat kidney: implication of transforming growth factor-β/Smad signaling pathway.

This study was designed to investigate the potential of extracellular adenosine 5'-triphosphate (ATP) via the P2 X 7 receptor to activate the renal fibrotic processes in rats. The present study demonstrates that administration of ATP rapidly activated transforming growth factor-β (TGF-β) to induce phosphorylation of Smad-2/3. Renal connective tissue growth factor (CTGF) and tissue inhibitor of metalloproteinase-1 (TIMP-1) mRNA and protein expressions were also increased following ATP administration. A decrease in TGF-β amount in serum as well as renal Smad-2/3 phosphorylation was noticed in animals pre-treated with the specific antagonist of P2 X 7 receptor, A 438,079. In addition, a significant reduction in mRNA and protein expression of CTGF and TIMP-1were also observed in the kidneys of those animals. Collectively, the current findings demonstrate that ATP has the ability to augment TGF-β-mediated Smad-2/3 phosphorylation and enhance the expression of the pro-fibrotic genes, CTGF and TIMP-1, an effect that is largely mediated via P2 X 7 receptor.

Pharmacokinetic-pharmacodynamic modeling of the active components of Shenkang injection in rats with chronic renal failure and its protective effect on damaged renal cells.

The study aimed to explore the pharmacokinetic and pharmacodynamic alterations of the active components of Shenkang injection (i.e. hydroxy saffron yellow pigment A [HSYA], tanshinol, rheum emodin, and astragaloside IV) in rats with chronic renal failure (CRF), and establish a pharmacokinetic-pharmacodynamic model (PK-PD model) in order to provide a scientific and theoretical basis for the rational clinical use of Shenkang injection. Sprague-Dawley (SD) rats were randomly divided into a normal group, model group, and Shenkang injection group. A rat model of CRF was induced by adenine gavage and then followed by drug administration via tail vein injection. Orbital blood was collected at different timepoints and the blood concentrations of the four active components were measured by UHPLC-Q-Orbitrap HRMS. Serum levels of creatinine (Scr), urea nitrogen (BUN), and uric acid (UA) were determined using an automatic biochemical analyzer. A PK-PD model was established, and DAS 3.2.6 software was used for model fitting as well as statistical analysis. TGF-β1 was utilized to induce normal rat kidney cells to construct a renal fibrosis model to investigate the protective effect of the pharmacological components on renal fibrosis. The pharmacokinetic analysis of hydroxy saffron yellow pigment A, tanshinol, rheum emodin, and astragaloside IV based on UHPLC-Q-Orbitrap HRMS was stable. The linear regression equations for the four active components were as follows: Y=0.031X+0.0091 (R2 =0.9986) for hydroxy saffron yellow pigment A, Y=0.0389X+0.164 (R2 =0.9979) for tanshinol, Y=0.0257X+0.0146 (R2 =0.9973) for rheum emodin, and Y=0.0763X+0.0139 (R2 =0.9993) for astragaloside IV, which indicated good linear relationships. The methodological investigation was stable, with the interday and intraday precision RSD <10%. Meanwhile, the recoveries ranged between 90% and 120%, in accordance with the requirements for in vivo analysis of drugs. Compared with the model group, the levels of Scr, BUN, and UA were significantly decreased after 20min in the Shenkang injection group (p<0.01). The PK-PD model showed that the four active components in the Shenkang injection group could fit well with the three effect measures (i.e. Scr, BUN, and UA), with the measured values similar to the predicted values. The cell model of renal fibrosis showed that the connective tissue growth factor and FN1 protein expression levels were significantly lower in the Shenkang injection group than those in the model group, and the cell fibrosis was improved. The established method for in vivo analysis of Shenkang injection was highly specific, with good separation of the components and simple operation. The total statistical moment could well integrate the pharmacokinetic parameters of the four active components. After treatment with Shenkang injection, all indexes in the administered group improved and showed significant inhibition of renal cell fibrosis in vitro. This study could provide scientific reference ideas for the clinical rational use of traditional Chinese medicine.

Loss of connective tissue growth factor expression promotes remodeling of the extracellular matrix and epithelial-to-mesenchymal transition in ovarian cancer (2294)

Loss of connective tissue growth factor expression promotes remodeling of the extracellular matrix and epithelial-to-mesenchymal transition in ovarian cancer (2294)

Estrogen inhibits the differentiation of fibroblasts induced by high stiffness matrix by enhancing DNMT1 expression

Introduction and hypothesisPelvic organ prolapse(POP) is a multifactorial connective tissue disorder caused by damage to the supporting structures of the pelvic floor. Evidence from several studies suggests that anterior vaginal wall stiffness is higher in patients with POP, but the mechanisms involved remain unknown. MethodsTissue from the anterior vaginal wall of patients with POP or other benign diseases was obtained. The modulus of elasticity of the anterior vaginal wall was measured using a microindenter. Cells were cultured in vitro on acrylamide gels of different stiffness and treated with DNMT1 inhibitor, microtubule polymerisation inhibitor and estrogen. Western blot or immunohistochemical staining was performed to detect DNA Methyltransferase 1, α-smooth muscle actin(α-SMA) expression, and connective tissue growth factor(CTGF) expression. ConclusionEstrogen can inhibit high stiffness matrix-induced fibroblast differentiation, by enhancing DNMT1 expression. This study may help to elucidate the complex crosstalk between fibroblasts and their surrounding matrix under healthy and pathological conditions and provide new insights into the options for material-related therapeutic applications.

Increased CTGF expression in alveolar epithelial cells by cyclic mechanical stretch: Its mechanism and the therapeutic effect of pirfenidone

The mechanisms of fibrosis onset and development remain to be elucidated. However, it has been reported that mechanical stretch promotes fibrosis in various organs and cells, and may be involved in the pathogenesis of pulmonary fibrosis. We demonstrated that ventilator-induced lung hyperextension stimulation in mice increased the expression of connective tissue growth factor (CTGF), a profibrotic cytokine, in lung tissue. Increased CTGF expression induced by cyclic mechanical stretch (CMS) was also observed in vitro using A549 human alveolar epithelial cells. Pathway analysis revealed that the induction of CTGF expression by CMS involved MEK phosphorylation. Furthermore, early growth response 1 (Egr-1) was identified as a transcription factor associated with CTGF expression. Finally, the antifibrotic drug pirfenidone significantly reduced CTGF expression, MEK phosphorylation, and Egr-1 levels induced by CMS. Thus, our results demonstrated that profibrotic cytokine CTGF induced by CMS may be a therapeutic target of pirfenidone.

Long noncoding RNA AI662270 promotes kidney fibrosis through enhancing METTL3-mediated m6 A modification of CTGF mRNA.

The sustained release of profibrotic cytokines, mainly transforming growth factor-β (TGF-β), leads to the occurrence of kidney fibrosis and chronic kidney disease (CKD). Connective tissue growth factor (CTGF) appears to be an alternative target to TGF-β for antifibrotic therapy in CKD. In this study, we found that long noncoding RNA AI662270 was significantly increased in various renal fibrosis models. In vivo, ectopic expression of AI662270 alone was sufficient to activate interstitial fibroblasts and drive kidney fibrosis, whereas inhibition of AI662270 blocked the activation of interstitial fibroblasts and ameliorated kidney fibrosis in various murine models. Mechanistic studies revealed that overexpression of AI662270 significantly increased CTGF product, which was required for the role of AI662270 in driving kidney fibrosis. Furthermore, AI662270 binds to the CTGF promoter and directly interacts with METTL3, the methyltransferase of RNA N6 -methyladenosine (m6 A) modification. Functionally, AI662270-mediated recruitment of METTL3 increased the m6 A methylation of CTGF mRNA and consequently enhanced CTGF mRNA stability. In conclusion, our results support that AI662270 promotes CTGF expression at the posttranscriptional stage by recruiting METTL3 to the CTGF promoter and depositing m6 A modifications on the nascent mRNA, thereby, uncovering a novel regulatory mechanism of CTGF in the pathogenesis of kidney fibrosis.

Isoliquiritigenin attenuates high glucose-induced proliferation, inflammation, and extracellular matrix deposition in glomerular mesangial cells by suppressing JAK2/STAT3 pathway.

To investigate the effect of isoliquiritigenin (ISL) on high glucose (HG)-induced glomerular mesangial cells (GMCs) proliferation, extracellular matrix (ECM) deposition and inflammation, and the underlying mechanisms. Mouse GMCs (SV40-MES-13) were cultured in HG medium, with or without ISL. The proliferation of GMCs was determined by MTT assay. The production of proinflammatory cytokines was detected by qRT-PCR and ELISA. The expression of connective tissue growth factor (CTGF), TGF-β1, collagen IV, and fibronectin was measured by qRT-PCR and western blot. The phosphorylation of JAK2 and STAT3 was examined by western blot. Next, JAK2 inhibitor AG490 was applied to HG-exposed GMCs. The levels of JAK2/STAT3 phosphorylation and pro-fibrotic markers were analyzed by western blot, and the secretion of TNF-α and IL-1β was evaluated by ELISA. GMCs were treated with HG, HG plus ISL or HG plus ISL, and recombinant IL-6 (rIL-6) which is a JAK2 activator. The levels of JAK2/STAT3 activation, ECM formation, and proinflammatory cytokines secretion were determined by western blot and ELISA, respectively. In mouse GMCs, ISL successfully repressed HG-induced hyperproliferation; production of TNF-α and IL-1β; expression of CTGF, TGF-β1, collagen IV, and fibronectin; and activation of JAK2/STAT3. Similar to ISL, AG490 was able to reverse the inflammation and ECM generation caused by HG. Moreover, rIL-6 impeded the amelioration of ISL on HG-induced adverse effects. Our study demonstrated that ISL displayed preventive effects on HG-exposed GMCs through inhibiting JAK2/STAT3 pathway and provided an insight into the application of ISL for diabetic nephropathy (DN) treatment.

Sodium hyaluronate promotes proliferation, autophagy, and migration of corneal epithelial cells by downregulating miR-18a in the course of corneal epithelial injury

Corneal epithelium can resist the invasion of external pathogenic factors to protect the eye from external pathogens. Sodium hyaluronate (SH) has been confirmed to promote corneal epithelial wound healing. However, the mechanism by which SH protects against corneal epithelial injury (CEI) is not fully understood. CEI model mice were made by scratching the mouse corneal epithelium, and in vitro model of CEI were constructed via curettage of corneal epithelium or ultraviolet radiation. The pathologic structure and level of connective tissue growth factor (CTGF) expression were confirmed by Hematoxylin and Eosin staining and immunohistochemistry. CTGF expression was detected by an IHC assay. The levels of CTGF, TGF-β, COLA1A, FN, LC3B, Beclin1, and P62 expression were monitored by RT-qPCR, ELISA, Western blotting or immunofluorescence staining. Cell proliferation was detected by the CCK-8 assay and EdU staining. Our results showed that SH could markedly upregulate CTGF expression and downregulate miR-18a expression in the CEI model mice. Additionally, SH could attenuate corneal epithelial tissue injury, and enhance the cell proliferation and autophagy pathways in the CEI model mice. Meanwhile, overexpression of miR-18a reversed the effect of SHs on cell proliferation and autophagy in CEI model mice. Moreover, our data showed that SH could induce the proliferation, autophagy, and migration of CEI model cells by downregulating miR-18a. Down-regulation of miR-18a plays a significant role in the ability of SH to promote corneal epithelial wound healing. Our results provide a theoretical basis for targeting miR-18a to promote corneal wound healing.

Endothelin-1 induces connective tissue growth factor expression in human lung fibroblasts by disrupting HDAC2/Sin3A/MeCP2 corepressor complex

BackgroundReduction of histone deacetylase (HDAC) 2 expression and activity may contribute to amplified inflammation in patients with severe asthma. Connective tissue growth factor (CTGF) is a key mediator of airway fibrosis in severe asthma. However, the role of the HDAC2/Sin3A/methyl-CpG-binding protein (MeCP) 2 corepressor complex in the regulation of CTGF expression in lung fibroblasts remains unclear.MethodsThe role of the HDAC2/Sin3A/MeCP2 corepressor complex in endothelin (ET)-1-stimulated CTGF production in human lung fibroblasts (WI-38) was investigated. We also evaluated the expression of HDAC2, Sin3A and MeCP2 in the lung of ovalbumin-induced airway fibrosis model.ResultsHDAC2 suppressed ET-1-induced CTGF expression in WI-38 cells. ET-1 treatment reduced HDAC2 activity and increased H3 acetylation in a time-dependent manner. Furthermore, overexpression of HDAC2 inhibited ET-1-induced H3 acetylation. Inhibition of c-Jun N-terminal kinase, extracellular signal-regulated kinase, or p38 attenuated ET-1-induced H3 acetylation by suppressing HDAC2 phosphorylation and reducing HDAC2 activity. Overexpression of both Sin3A and MeCP2 attenuated ET-1-induced CTGF expression and H3 acetylation. ET-1 induced the disruption of the HDAC2/Sin3A/MeCP2 corepressor complex and then prompted the dissociation of HDAC2, Sin3A, and MeCP2 from the CTGF promoter region. Overexpression of HDAC2, Sin3A, or MeCP2 attenuated ET-1-stimulated AP-1-luciferase activity. Moreover, Sin3A- or MeCP2-suppressed ET-1-induced H3 acetylation and AP-1-luciferase activity were reversed by transfection of HDAC2 siRNA. In an ovalbumin-induced airway fibrosis model, the protein levels of HDAC2 and Sin3A were lower than in the control group; however, no significant difference in MeCP2 expression was observed. The ratio of phospho-HDAC2/HDAC2 and H3 acetylation in the lung tissue were higher in this model than in the control group. Overall, without stimulation, the HDAC2/Sin3A/MeCP2 corepressor complex inhibits CTGF expression by regulating H3 deacetylation in the CTGF promoter region in human lung fibroblasts. With ET-1 stimulation, the HDAC2/Sin3A/MeCP2 corepressor complex is disrupted and dissociated from the CTGF promoter region; this is followed by AP-1 activation and the eventual initiation of CTGF production.ConclusionsThe HDAC2/Sin3A/MeCP2 corepressor complex is an endogenous inhibitor of CTGF in lung fibroblasts. Additionally, HDAC2 and Sin3A may be of greater importance than MeCP2 in the pathogenesis of airway fibrosis.