CTGF mRNA Levels Research Articles

Y-27632 enables long-term expansion of mouse submandibular gland epithelial cells via inactivation of TGF-β1/CTGF/p38 and ROCK2/JNK signaling pathway

ObjectivesThis study aimed to investigate the effects of Y-27632 on the long-term maintainence of mouse submandibular epithelial cells (SG-Epis) in vitro and to elucidate the underlying mechanisms. MethodsThe role of the Rho-associated kinase (ROCK) inhibitor Y-27632 in maintaining SG-Epis and its underlying mechanisms were evaluated by examining the in vitro expansion of mouse SG-Epis. Changes in key cellular characteristics, such as proliferation, long-term expansion, and mRNA and protein expression, were assessed in the presence or absence of Y-27632. ResultsTreatment with Y-27632 significantly enhanced the proliferative potential of SG-Epis, preserving Krt8 and Krt14 expression over 17 passages. In the absence of Y-27632, SG-Epis lost their epithelial morphology. However, Y-27632 treatment maintained the epithelial morphology and downregulated mRNA levels of Tgf-β1, Ctgf, and Rock2. Treatment with TGF-β1 indicated that TGF-β/CTGF/p38 signaling is responsible for the maintenance of SG-Epis, while RNA interference studies revealed that ROCK2/c-Jun N-terminal kinase (JNK) signaling is also crucial for SG-Epis proliferation and maintenance. ConclusionsThe TGF-β1/CTGF/p38 and ROCK2/JNK signaling pathways are responsible for SG-Epis proliferation, and Y-27632 treatment effectively inactivates these pathways, enabling long-term in vitro maintenance of SG-Epis. The culture method utilizing Y-27632 provides an effective approach for the in vitro expansion of SG-Epis.

Loss of Myeloid-Derived Growth Factor Leads to Increased Fibrosis in Mice After Myocardial Infarction

To investigate the effect of the loss of myeloid-derived growth factor (Mydgf) on the transformation of cardiac fibroblasts into myofibroblasts after myocardial infarction (MI). Two adult mouse groups, including a wild-type (WT) group and another group with Mydgf knockout (Mydgf-KO), were examined in the study. The mice in these two groups were tested for their cardiac function by measuring left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) (n=10). Quantitative real-time PCR (qRT-PCR) (n=3) was performed to determine the mRNA expression levels of myofibroblast markers, including α-smooth muscle actin (α-SMA), periostin (postn), type Ⅷ collagen (col8al), and connective tissue growth factor (ctgf). Western blot (n=3) was performed to verify the protein expression levels of α-SMA. MI modeling was performed on the WT and the Mydgf-KO mice. Postoperative LVEF and LVFS (n=10) were then measured. The hearts were harvested and Masson staining was performed to determine the infarcted area (n=10). The heart samples of Mydgf-KO and WT mice were collected at d 7 and d 14 after MI, respectively, to verify the expression of myofibroblast markers (n=3). Compared with WT mice, LVEF and LVFS in adult Mydgf-KO mice showed no significant changes (all P>0.05). However, the mRNA levels of α-SMA and postn were upregulated, and α-SMA protein expression was also increased (all P<0.05). After MI, compared with WT mice, LVEF and LVFS in Mydgf-KO mice decreased, and the infarcted area increased significantly (all P<0.05). Furthermore, mRNA levels of α-SMA, col8al, postn, and ctgf were increased in Mydgf-KO mice. In addition, the α-SMA protein expression level was upregulated and α-SMA-positive fibroblasts were increased (P<0.05). Mydgf deletion promotes the transformation of cardiac fibroblasts into myofibroblasts and aggravates myocardial fibrosis after MI.

Integrin-Linked Kinase in the Development of Gastric Tumors Induced by Helicobacter pylori: Regulation and Prevention Potential.

Integrin-linked kinase (ILK) is crucial in solid tumors by regulating the Hippo-Yes-associated protein 1 (YAP) pathway. This study aimed to uncover how Helicobacter pylori influences ILK levels and its role in regulating YAP during H. pylori-induced gastric cancer. GES-1 cells with stable Ilk knockdown and overexpression and a mouse carcinogenesis model for H. pylori infection were constructed. And ILK, the phosphorylated mammalian STE20-like protein kinase 1 (MST1), large tumor suppressor 1 (LATS1; S909, T1079), and YAP (S109, S127) were detected in cells, and mice by western blotting, as well as fluorescence intensity of YAP were assayed by immunofluorescence. YAP downstream genes Igfbp4 and Ctgf, the pathological changes and tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), interleukin-1beta (IL-1β), and nitric oxide (NO) levels in mice gastric tissues were detected by real-time PCR, H&E, and ELISA assays. In this study, stable Ilk knockdown cells exhibited significantly higher phosphorylated levels of MST1, LATS1, and YAP, as well as increased YAP in the nuclei of GES-1 cells. Conversely, cells with Ilk overexpression showed opposite results. H. pylori infection led to decreased ILK levels in gastric epithelial cells but increased ILK levels in gastric cancer cell lines (MGC803, SGC7901) and gastric cancer tissues in mice. Treatment with the ILK inhibitor OST-T315 elevated the phosphorylated MST, LATS1, and YAP levels, and inhibited the mRNA levels of Igfbp4 and Ctgf at 44, 48 week-aged mice. OST-T315 also reduced the release of TNF-α, IL-6, IL-1β, and NO, as well as the progression of gastric cancer caused by H. pylori and N-Nitroso-N-methylurea (NMU) treatment. Upon initiation of gastric tumorigenesis signals, H. pylori increases ILK levels and suppresses Hippo signaling, thereby promoting YAP activation and gastric cancer progression. ILK can serve as a potential prevention target to impede H. pylori-induced gastric cancer.

Tubuloside B, a major constituent of Cistanche deserticola, inhibits migration of hepatocellular carcinoma by inhibiting Hippo-YAP pathway

BackgroundStudies have shown that phenylethanoid glycosides (PhGs) have multiple pharmacological effects such as anti-inflammatory, hepatoprotective or neuroprotective functions, whereas their anti-tumor effects are rarely studied. Tubuloside B (Tub B) is a PhG isolated from Cistanche deserticola, a traditional Chinese medicine. To date, there is a lack of comprehensive research regarding the biological activity of Tub B. PurposeThe subject of the current study was to investigate the anti-hepatocellular carcinoma (HCC) cell activity and the underlying mechanism of Tub B. MethodsWe evaluated the in vitro anti-migratory effect of Tub B by scratch and transwell assays. RNA-seq was employed to identify the differential genes by Tub B. Besides, the functional mechanism of Tub B was investigated by distinct molecular biology techniques including immunofluorescent staining, quantitative PCR, as well as western blot analysis. Subsequently, we utilized Hep3B cells for in vivo metastasis assays through spleen injection and evaluated the anti-migratory effect of Tub B in hepatocellular carcinoma (HCC). ResultsTub B exhibited in vitro and in vivo inhibition of HCC cell migration. Tub B decreased the expression of transcriptional target genes downstream of the Hippo pathway, including CTGF, CYR61, and N-cadherin as determined by RNA-seq. Furthermore, mechanistic studies confirmed that Tub B increased phosphorylation of YAP at S127, which contributes to YAP cytoplasmic localization. Additionally, overexpression of YAP abrogated Tub B-induced inhibition of HCC migration and the mRNA levels of CTGF, CYR61, and N-cadherin. ConclusionsTaken together, these results illustrated that Tub B demonstrated great potential in inhibiting migration of HCC, and a portion of its impact can be attributed to the modulation of the Hippo-YAP pathway.

The XPO1 inhibitor selinexor ameliorates bleomycin-induced pulmonary fibrosis in mice via GBP5/NLRP3 inflammasome signaling

Pulmonary fibrosis is an irreversible and progressive lung disease with limited treatments available. Selinexor (Sel), an orally available, small-molecule, selective inhibitor of XPO1, exhibits notable antitumor, anti-inflammatory and antiviral activities. However, its potential role in treating pulmonary fibrosis is unknown. C57BL/6J mice were used to establish a pulmonary fibrosis model by intratracheal administration of bleomycin (BLM). Subsequently, Sel was administered intraperitoneally. Our data demonstrated that Sel administration ameliorated BLM-induced pulmonary fibrosis by increasing mouse body weights; reducing H&E staining, Masson staining scores, and shadows in mouse lung computed tomography (CT) images, decreasing the total cell and neutrophil counts in the lung and bronchoalveolar lavage fluid (BALF); and decreasing the levels of TGF-β1. We next confirmed that Sel reduced the deposition of extracellular matrix (ECM) components in the lungs of BLM-induced pulmonary fibrosis mice. We showed that collagen I, alpha-smooth muscle actin (α-SMA), and hydroxyproline levels and the mRNA levels of Col1a1, Eln, Fn1, Ctgf, and Fgf2 were reduced. Mechanistically, tandem mass tags (TMT)- based quantitative proteomics analysis revealed a significant increase in GBP5 in the lungs of BLM mice but a decrease in that of BLM + Sel mice; this phenomenon was confirmed by western blotting and RT-qPCR. NLRP3 inflammasome signaling was significantly enriched in both the BLM group and BLM + Sel group based on GO and KEGG analyses of differentially expressed proteins between the groups. Furthermore, Sel reduced the expression of NLRP3, cleaved caspase 1, and ASC in vivo and in vitro, and decreased the levels of IL-1β, IL-18, and IFN-r in lung tissue and BALF. SiRNA-GBP5 inhibited NLRP3 signaling in vitro, and overexpression of GBP5 inhibited the protective effect of Sel against BLM-induced cellular injury. Taken together, our findings indicate that Sel ameliorates BLM-induced pulmonary fibrosis by targeting GBP5 via NLRP3 inflammasome signaling. Thus, the XPO1 inhibitor – Sel might be a potential therapeutic agent for pulmonary fibrosis.

Superoxide dismutase secreting Bacillus amyloliquefaciens spores attenuate pulmonary fibrosis

Superoxide dismutase (SOD) can convert active oxygen to oxygen or hydrogen peroxide, and recent research has suggested that it can protect against lung damage and fibrosis. Clinical applications based on SOD remain limited however due to costs and low stability. We here investigated a potential new therapeutic delivery system for this enzyme in the form of SOD-overexpressing Bacillus amyloliquefaciens spores which we introduced into a bleomycin-induced pulmonary fibrosis mouse model. This treatment significantly alleviated the disease, as quantified using a hydroxyproline assay, at 107 colony forming unit (CFU) of Bacillus spores per day. Exposure of the mice to the spores was further found to decrease the lung mRNA levels of CTGF, Col1a1, α-SMA, TGF-β, TNF-α, and IL-6, and the protein levels of TGF-β, Smad2/3, αSMA and Col1a1, all major indicators of pulmonary fibrosis. Survival benefits, and reduced byproducts of lipid peroxidase such as malondialdehyde and 4-hydroxynen, were also noted in the treated animals. The beneficial effects of these Bacillus spores on pulmonary fibrosis were further found to be greater than the equivalent free SOD concentration. Immunofluorescence staining of primary pulmonary fibroblasts extracted from the bleomycin-induced model showed decreased αSMA expression following the in vivo treatment with SOD-overexpressing Bacillus. Our treatment approach SOD through Bacillus spores shows beneficial effects against pulmonary fibrosis, combined with the suppression of the SMAD/TGF-β pathway, suggesting that it is an effective novel delivery route for antioxidants.

The Hippo signaling pathway contributes to the 2,5-Hexadion-induced apoptosis of ovarian granulosa cells

Although n-hexane can induce ovarian damage by inducing ovarian granulosa cell (GC) apoptosis, the mechanism underlying this induction of apoptosis has not been fully elucidated. In this study, rat ovarian GCs were exposed to different concentrations of 2,5-hexanedione (2,5-HD) (the main metabolite of n-hexane) in vitro to observe apoptosis, and the mechanism was further explored via mRNA microarray analysis. Hoechst 33258 staining and flow cytometry suggested that the apoptosis rate of ovarian GC apoptosis was significantly increased in the 2,5-HD-treated group. Subsequently, microarray analysis revealed that a total of 5677 mRNAs were differentially expressed, and further GO and KEGG analyses revealed that the differentially expressed genes were significantly enriched in many signaling pathways, including the Hippo pathway. A total of 7 differentially expressed genes that function upstream of the Hippo signaling pathway (Nf2, Wwc1, Ajuba, Llgl1, Dlg3, Rassf6 and Rassf1) were selected to confirm the microarray results by qRT-PCR, and the expression of these genes did change. Subsequently, the expression of key effector genes (Yap1, Mst1 and Lats1) and target genes (Ctgf and Puma) of the Hippo signaling was measured, and the results suggested that the mRNA and protein levels of Yap1, Mst1, Lats1, and Ctgf were significantly decreased while those of Puma were significantly increased after 2,5-HD treatment. Further CO-IP analysis suggested that the interaction between YAP1 and TEAD was significantly reduced after 2,5-HD treatment, while the interaction between YAP1 and P73 was not affected. In summary, during the 2,5-HD-induced apoptosis of ovarian GCs, the Hippo signaling pathway is inhibited, and downregulation of the pro-proliferation gene Ctgf and upregulated of the pro-apoptosis gene Puma are important. Decreased Ctgf expression was associated with decreased binding of YAP1 to TEAD. However, increased PUMA expression was not associated with YAP1 binding to P73.

Hydnocarpin inhibits malignant progression of triple negative breast cancer via CNOT4-mediated ubiquitination and degradation of YAP

This study aims to investigate the effect and mechanism of hydnocarpin(HC) in treating triple negative breast cancer(TNBC). Cell counting kit-8(CCK-8), xCELLigence real-time cellular analysis(RTCA), and colony formation assay were employed to determine the effects of HC on the proliferation of two TNBC cell lines: MDA-MB-231 and MDA-MB-436. The effects of HC on the migration and invasion of TNBC cells were detected by high-content analysis, wound-healing assay, and Transwell assay. The changes in the epithelial-mesenchymal transition(EMT) and the expression of invasion-and migration-associated proteins [E-cadherin, vimentin, Snail, matrix metalloproteinase-2(MMP-2), and MMP-9] were detected by Western blot. Western blot and RT-qPCR were employed to determine the protein and mRNA levels of Yes-associated protein(YAP) and downstream targets(CTGF and Cyr61). TNBC cells were transfected with Flag-YAP for the overexpression of YAP, and the role of YAP as a key target for HC to inhibit TNBC malignant progression was examined by CCK-8 assay, Transwell assay, and wound-healing assay. The pathway of HC-induced YAP degradation was detected by the co-treatment of proteasome inhibitor with HC and ubiquitination assay. The binding of HC to YAP and the E3 ubiquitin ligase Ccr4-not transcription complex subunit 4(CNOT4) was detected by microscale thermophoresis(MST) assay and drug affinity responsive target stability(DARTS) assay. The results showed that HC significantly inhibited the proliferation, colony formation, invasion, and EMT of TNBC cells. HC down-regulated the protein and mRNA levels of CTGF and Cyr61. HC down-regulated the total protein level of YAP, while it had no effect on the mRNA level of YAP. The overexpression of YAP antagonized the inhibitory effects of HC on the proliferation, migration, and invasion of TNBC cells. HC promoted the degradation of YAP through the proteasome pathway and up-regulated the ubiquitination level of YAP. The results of MST and DARTS demonstrated direct binding between HC, YAP, and CNOT4. The above results indicated that HC inhibited the malignant progression of TNBC via CNOT4-mediated degradation and ubiquitination of YAP.

The ovarian-related effects of polystyrene nanoplastics on human ovarian granulosa cells and female mice

Nanoplastics (NPs) have recently emerged in the context of global plastic pollution. They may be more toxic than macroplastics litter and microplastic fragments due to its abundances, tiny sizes, and cellular accessibility. The female reproductive toxicity of NPs has been widely documented for aquatic animals, but their effects and underlying mechanisms remain poorly understood in mammals. This study aimed to explore the effects of NPs on female reproduction using human ovarian granulosa cells (GCs) and female mice. The accumulation of polystyrene NPs (PS-NPs) in human granulosa-like tumor cells (KGN cells) and the ovaries of female Balb/c mice were evaluated by exposure to fluorescent PS-NPs. Proliferation and apoptosis, reactive oxygen species (ROS), and Hippo signaling pathway-related factors were analyzed in KGN cells. In addition, fertility rate, litter size, ovarian weight and microstructure, follicle development, serum level of anti-Mullerian hormone, and apoptosis in ovaries were examined in female mice. Here, the PS-NPs can penetrate the KGN cells and accumulate in the ovaries. In vitro, 100 μg/ml PS-NPs inhibited proliferation, induced apoptosis, accumulated ROS, activated three key regulators of the Hippo signaling pathway (MST1, LATS1, and YAP1), and downregulated the mRNA levels of CTGF and Cyr61 in KGN cells. Furthermore, salidroside, an antioxidative compound extracted from Rhodiola rosea, alleviated the damage of PS-NPs to KGN and inhibited the activation of the Hippo signal pathway. In vivo, exposure to 1 mg/day PS-NPs resulted in decreased fertility, abnormal ovarian function, and increased ovarian apoptosis in female mice. Overall, our data suggest that PS-NPs cause granulosa cell apoptosis and affect ovarian functions, leading to reduced fertility in female mice, by inducing oxidative stress and dysregulating the Hippo pathway.

Small-Molecule Cyanamide Pan-TEAD·YAP1 Covalent Antagonists.

Transcriptional enhanced associate domains (TEADs) are transcription factors that bind to cotranscriptional activators like the yes-associated protein (YAP) or its paralog transcriptional coactivator with a PDZ-binding motif (TAZ). TEAD·YAP/TAZ target genes are involved in tissue and immune homeostasis, organ size control, tumor growth, and metastasis. Here, we report isoindoline and octahydroisoindole small molecules with a cyanamide electrophile that forms a covalent bond with a conserved cysteine in the TEAD palmitate-binding cavity. Time- and concentration-dependent studies against TEAD1-4 yielded second-order rate constants kinact/KI greater than 100 M-1 s-1. Compounds inhibited YAP1 binding to TEADs with submicromolar IC50 values. Cocrystal structures with TEAD2 enabled structure-activity relationship studies. In mammalian cells, compounds suppressed CTGF mRNA levels and inhibited TEAD1-4 transcriptional activity with submicromolar IC50 values. Inhibition of TEAD binding to YAP1 in mammalian cells was also observed. Several compounds inhibited the cell viability of sarcoma, hepatocellular carcinoma, glioblastoma, and breast cancer cells with single-digit micromolar IC50 values.

Design, synthesis and biological evaluation studies of novel anti-fibrosis agents bearing sulfoxide moiety

Fibrosis, a chronic disease with high morbidity and mortality, is mainly characterized by excessive accumulation of extracellular matrix (ECM). At present, pathogenesis of fibrosis is incompletely understood, and there is an urgent need to develop safe and effective drugs. In this study, we designed and synthesized a series of novel small-molecule compounds through structural modification and fragment hybridization. Among them, a potential anti-fibrosis drug compd.1 was founded to be able to dose-dependently down-regulate ACTA2 and CTGF mRNA levels in human hepatic stellate cells (LX-2) treated with TGF-β. In addition, compd.1 significantly improved the bridging fibrosis and collagen content in the CCl4-induced liver fibrosis mice model. Moreover, compd.1 reduced lung inflammation and fibrotic area in bleomycin-induced pulmonary fibrosis mice model. These findings suggested that compd.1 is a promising candidate for further anti-fibrosis researches, and extended chemical space might help us to explore better anti-fibrosis drug.

VGLL4 inhibits YAP1/TEAD signaling to suppress the epidermal squamous cell carcinoma cancer phenotype.

Epidermal squamous cell carcinoma (SCC) develops in response to ultraviolet light exposure and is among the most common cancers. The transglutaminase 2 cancer cell survival protein stimulates the activity of the YAP1/TEAD transcription complex to drive the expression of genes that promote aggressive epidermal SCC cell invasion, migration, and tumor formation. Therefore, we are interested in mechanisms that may inhibit these events. Vestigial-like protein-4 (VGLL4) is a transcription cofactor/tumor suppressor that inhibits several pro-cancer pathways including YAP1 signaling. Our present studies show that VGLL4 inhibits YAP1/TEAD-dependent transcription to reduce the expression of YAP1 target genes (CCND1, CYR61, and CTGF) and pro-cancer collagen genes (COL1A2and COL3A1). We further show that loss of these YAP1 regulated genes is required for VGLL4 suppression of the cancer cell phenotype, as forced CCND1 or COL1A2 expression partially restores the aggressive cancer phenotype in VGLL4 expressing cells. Consistent with these findings, VGLL4 expression reduces tumor formation, and this is associated with reduced CCND1, CYR61, CTGF, COL1A2, and COL1A3 mRNA and protein levels, and reduced EMT marker expression. These findings indicate that VGLL4 suppresses the malignant epidermal SCC cancer phenotype by inhibiting YAP1/TEAD-dependent pro-cancer signaling.

Mo1290 THE EFFECT OF PIRFENIDONE FOR ESOPHAGEAL STRICTURE AFTER CIRCUMFEREENTIAL ESOPHAGEAL ENDOSCOPIC SUBMUCOSAL DISSECTION IN A PORCINE MODEL

Esophageal stricture inevitably comes up after endoscopic submucosal dissection (ESD) for circumferential esophageal cancer. The effect of current preventive treatments has not been sufficient, although several studies have been reported in multifaceted methods. We focused on Pirfenidone, which is an oral antifibrotic drug commonly used for idiopathic pulmonary fibrosis. Our aim was to investigate the effect of Pirfenidone for preventing esophageal stricture after circumferential esophageal ESD in a porcine model. Eight four-months-old female three-way crossed (LWD) pigs were used for this study. Eight pigs underwent a 5cm-long circumferential ESD under general anesthesia on day 1. Two pigs were used as a control group. Pirfenidone was orally administrated (800mg/day) to 2 pigs and (1200mg/day) to 4 pigs for fourteen consecutive days. Biological tissues were sampled by using forceps on day 1, day 8 and day 15. The pigs were euthanized on day 15, followed by evaluations on esophageal stricture macroscopically and microscopically, and the expression of genes closely associated with the development of fibrosis. The esophageal contraction rate (%) was calculated in extracted specimens macroscopically. The contraction rate (%) was defined as 100 (A - B)/A, where A is the mean value of oral diameter and anal diameter each 2cm from the edge after ESD and B is the narrowest diameter of esophagus. Esophageal fibrosis was identified by Masson’s Trichrome staining microscopically. The expression of genes was evaluated on the messenger RNA (mRNA) levels of IL-6, IL-1β, TNFα, TGF-β1, CTGF, FGF2, EGF, collagen1 and αSMA by Real-Time RT-PCR method. The contraction rate was 61.1% in the control group, 67.8% in Pirfenidone (800mg/day) and 58.1% in Pirfenidone (1200mg/day) groups. Pirfenidone didn’t reduce the stricture rate. The pathological findings or the expression of genes showed no significant differences. In the control group, the expression levels of TGF-β1 reached the maximum value on day 8 and decreased on day15. Whereas the expression levels of CTGF, FGF2, EGF, collagen1 and αSMA escalated stepwise. We couldn’t demonstrate the effect of Pirfenidone for esophageal stricture after circumferential esophageal ESD in a porcine model. However, results of this study might include some beneficial information for our further study.

CREBH knockout accelerates hepatic fibrosis in mouse models of diet-induced nonalcoholic fatty liver disease

AimsThe primary focus of this study was to explore the effects of cyclic AMP response element-binding protein H (CREBH) on the development of nonalcoholic fatty liver disease (NAFLD). Materials and methodsCREBH knockout (KO) and wildtype (WT) mice were averagely divided into a methionine and choline-deficient (MCD) or high fat (HF) diet group and respective chow diet (CD) groups. Mice were sacrificed after 4-week treatment for MCD model and 24-week treatment for HF model. Key findingsCharacteristics of nonalcoholic steatohepatitis-related liver fibrosis in KO-MCD/HF group were verified by hepatic histological analyses. Compared with WT-MCD/HF group, levels of plasma ALT and hepatic hydroxyproline increased in KO-MCD/HF group. Significantly higher levels of MCP-1, αSMA, Desmin, COL-1, TIMP-1, TGF-β1, TGF-β2 were found while MMP-9 and FGF21 mRNA levels decreased in KO-MCD/HF group. There was also a distinct difference of mRNA levels of TNFα, CTGF and CCND1 in KO-HF group compared with controls. Protein levels of MCP-1, BAX, αSMA, COL-1, TGF-β1 and SMAD2/3 significantly increased in KO-MCD/HF group and CCND1 was also upregulated in KO-HF group compared to their counterparts. SignificanceCREBH knockout may primarily regulate the TGF-β1 signaling pathway via TGF-β2 and FGF21 resulting in more severe inflammation and fibrosis in NAFLD.

Effects of intravitreal connective tissue growth factor neutralizing antibody on choroidal neovascular membrane-associated subretinal fibrosis

Connective tissue growth factor (CTGF) plays an essential role in the regulation of extracellular matrix proteins and pro-fibrotic and angiogenic factors. This experimental research was conducted to evaluate if CTGF is elevated after induction of a choroidal neovascular membrane (CNVM) and whether intravitreal anti-CTGF without and with intravitreal bevacizumab (IVB) may have any effect on the CNVM associated sub-retinal fibrosis. In adherence to ARRIVE guidelines, CNVM was induced by laser spots in the right eye retinas of ninety-four pigmented rats. Quantitative real-time reverse transcription PCR (qRT-PCR) and western-blot analysis were performed on sclerochoroidal tissues of forty-four rats before and at different time intervals after laser application. The remaining fifty rats were randomly divided into five groups after laser application. Group A received intravitreal injection of 2 μl of the 50 μg/ml anti-CTGF. In group B, intravitreal injection of 2 μl of 25 mg/ml bevacizumab was performed. Group C received 1 μl intravitreal anti-CTGF and 1 μl IVB. Group D did not receive any intravitreal injection as the control group. In group E, intravitreal injection of 2 μl of nonspecific purified mouse IgG antibody was performed as the placebo group. After two weeks, double immunohistochemistry was performed by isolectin B4 and anti-collagen type1 on the sclerochoroidal flat-mounts. Masked measurement of the fluorescent images of the CNVM and CNVM associated sub-retinal fibrosis areas was performed using the image J software. Ctgf mRNA and CTGF protein levels increased to the maximum level in 24 h after laser application and remained higher than the control level up to the 14th day for the Ctgf mRNA and up to the 7th day for the CTGF protein level. Means of CNVM associated sub-retinal fibrosis areas in three treatment groups (A, B and C) were significantly less than the control (D) and placebo (E) groups (P < 0.001, <0.05, <0.001 respectively). For groups A and C, mean CNVM associated sub-retinal fibrosis areas were also significantly less than group B (P < 0.05 and < 0.01, respectively). In conclusion, this study showed significant reduction of the CNVM associated sub-retinal fibrosis via inhibition of the CTGF which mediates the final steps of fibrosis in various inflammatory and angiogenic pathways.

OR-011 Effect of Aerobic and Resistance Exercise on TGF-β1/Smad3 Signal Pathway and Collagen in Skeletal Muscle of Aging Mice

Objective &#x0D; The purpose of this study was to investigate the effects of TGF-β 1/Smad3 signaling pathway and its downstream factor CTGF in collagen deposition and its molecular mechanism. And then it explored further the effect of exercise on the TGF-β1/Smad3 signaling pathway and collagen deposition in skeletal muscle. Therefore, it is expected to provide alternative exercise intervention approaches for skeletal muscle diseases, which are caused by age-related changes of collagen, and to provide new research perspectives for skeletal muscle satellite cell activation and skeletal muscle regeneration.&#x0D; Methods 21 male BALB/c mice were normally raised from 4 weeks to 36 weeks under standard conditions. The mice was divided randomly into three groups, including: group C, the quiet control group; Group A, the aerobics training group, received nine weeks of treadmill training without loading; And the group R, the resistance training group, received nine weeks of climbing training with loading. The body weight and limb grip of the mice were measured on regularly during the experiment. After 24 hours of the last intervention experiment, the mice were weighed and then executed by dislocating the cervical spine. The quadriceps were taken. Real-time PCR technology was used to detect the mRNA levels of TGF-β1, TβR I, Smad 3, CTGF, Pax7, COL1 and COL3. Western blotting technique was used to detect the protein levels of TGF-β1, Smad3, P-Smad3 , CTGF, COL1, COL3, Pax7 and MyoD . The deposition of collagen in the quadriceps muscle tissue of mice was detected by Sirius red staining. And the localization and expressions of COL1 and Pax7 in the quadriceps of mice were demonstrated by immunohistochemistry and immunofluorescence technology respectively. &#x0D; Results (1) Compared with group C, the weight of mice in group A was significantly increased (P&lt;0.05), and the ratio of the wet weight of the quadriceps and the body weight of the mice increased significantly (P&lt;0.05), while there was no significant change on the limbs relative grip strength. Compared with group C, the body weight of mice in group R showed a certain degree of increase but no significant difference, the ratio between the wet weight of the quadriceps and the body weight of the mice was significantly increased (P&lt;0.01), and the limbs relative grip strength was significantly increased (P&lt;0.05).&#x0D; (2) Compared with group C, there was no significant difference in the mRNA and protein expression of COL1 and COL3 in the quadriceps of mice in group A, and there was no significant change in collagen volume fraction. Compared with group C, the mRNA and protein expression of COL1 and COL3 of the quadriceps in group R were significantly decreased (P&lt;0.05), and collagen volume fraction significantly reduced (P&lt;0.05), and collagen deposition decreased.&#x0D; (3) Compared with group C, the mRNA level of CTGF and the protein level of TGF-β1 and CTGF in quadriceps tissues of mice in group A were significantly decreased (P&lt;0.05). While the protein levels of Smad3 and p-Smad3 and the ratio of Smad3 and p-Smad3 had no significant change. Group R is compared with group C, the mRNA level of TGF-β1, TβR I and CTGF in quadriceps were significantly decreased (P&lt;0.05); the mRNA levels of Smad3 and the protein levels of TGF-β1 and p-Smad3 were significantly decreased (P&lt;0.01); and the protein levels of Smad3 and CTGF and the ratio of Smad3 and p-Smad3 were also significantly decreased (P&lt;0.05).&#x0D; (4) Compared with group C, the mRNA and protein expression of Pax7, and the protein expression of MyoD in the quadriceps of group A showed no significant difference. But group R is compared to group C, the expression of Pax7 mRNA in the quadriceps was significantly increased (P&lt;0.01), and the expression of Pax7 protein was also significantly increased (P&lt;0.05), while the protein expression of MyoD did not change significantly. Compared with group A, the mRNA levels of Pax7 in the quadriceps of the R group was significantly increased (P&lt;0.05), but the protein expression of Pax7 and MyoD showed no significant change.&#x0D; Conclusions &#x0D; (1) Through 9 week resistance or aerobic exercise training, skeletal muscle mass index in mice increased significantly; and the resistance exercise training can improve the limbs relative grip strength to prevent sacorpenia.&#x0D; (2) 9 week resistance exercise training can inhibit TGF-β1/Smad3 signaling pathway, affect the gene expression of COL1 and COL3, inhibit collagen synthesis, and improve collagen deposition.&#x0D; (3) 9 weeks of resistance exercise training can effectively promote Pax7 gene expression, activate skeletal muscle satellite cells and promote its proliferation.&#x0D; (4) The effect of 9 week of resistance exercise training on the improvement of skeletal muscle mass, strength, collagen deposition and the activation of satellite cells was significantly better than that of aerobic exercise.

Effects of crystalline silica with different exposure patterns on lung fibrosis in rats

Objective: To investigate the effects of crystalline silica with different exposure patterns on lung fibrosis in rats. Methods: A total of 20 adult male Wistar rats were randomly divided into 4 groups consisting of five animals each and received intratracheal instillation of sterile saline or silica suspension in different patterns: saline once at day 0, saline once/week, crystalline silica 50 mg at day 0, crystalline silica 6.25 mg/week. The rats were sacrificed at 8 weeks. The lung tissues were collected for pathological analyses, and determining mRNA and protein levels of related fibrogenic molecules. Results: The collagen deposition induced by crystalline silica in lung tissues were increased. The mRNA levels of IL-1β and Col I in group c were significantly elevated than those in group saline once at day 0 (all P<0.05). Compared with group saline once/week, the mRNA levels of IL-1β, TGF-β, Col I, Col III and CTGF were significantly increased in group crystalline silica 6.25 mg/week (P<0.05) . The mRNA levels of Col I and CTGF were significantly increased in group crystalline silica 6.25 mg/week in comparison with those in group crystalline silica 50 mg at day 0 (P<0.05) . Conclusion: Given the same cumulative dose of crystalline silica, multiple exposures were likely to induce more severe lung fibrosis.

Anti-Schistosomiasis Liver Fibrosis Effect of Corilagin Through Interacting the IL-13/miR-21/smads Signaling Pathway &lt;i&gt; in Vitro&lt;/i&gt; and &lt;i&gt; in Vivo&lt;/i&gt;

Background: This study was to investigate the anti-fibrosis effect of corilagin on schistosomiasis liver fibrosis through interacting IL-13/miR-21/smads signaling pathway. Methods: The cellular models were established with LX2 cells and primary mouse hepatic stellate cells stimulated by IL-13. MiR-21 was regulated up or down by lentiviral vector transfection, and smad1 and smad2 were transfected by siRNA for down-regulation. Mice were infected by schistosome cercariae as animal model. The real-time PCR was employed to detect mRNA levels of miR-21, smad7 and CTGF, and western blot was used to examine protein levels of smad7, CTGF, smad1, p-smad1, smad2, p-smad2, p-smad2/3, smad4, TβR Ⅰ and α-SMA. IL-13 and SCAg in serum were measured by ELISA. Immunohistochemistry was used to test the expression of smad7. The liver tissue was tested for hematoxylin-eosin and masson staining. Findings: No matter in normal, miR-21 up- or down-regulated LX2 cells, corilagin could inhibit the mRNA level of miR-21 and CTGF and promote smad7 mRNA level (P < 0.01 or 0.05). Meanwhile, corilagin could significantly reduce the protein level of CTGF, p-smad1, p-smad2, psmad2/3, smad4, TβRⅠand α-SMA and promote smad7 protein level (P <0.05 or 0.01). Corilagin could decrease serum IL-13 and SCAg level (P < 0.01 or 0.05), and also reduce the degree of liver fibrosis in pathological manifestations. Interpretation: Corilagin could inhibit schistosomiasis-induced hepatic fibrosis through the potential drug intervention targets in IL-13/miR21/smads pathway, which might serve as an anti-fibrosis agent for treating schistosomiasis liver fibrosis. Funding Statement: This study was supported by the National Natural Science Foundation of China (81371840), Hubei Province Health and Family Planning Scientific Research Project (WJ2017Q021), Hubei Provincial Natural Science Foundation of China (2017CFB471), the Fundamental Research Funds for the Central Universities (2017KFYXJJ238). Declaration of Interests: The authors have no financial conflicts to declare. Ethics Approval Statement: The study was reviewed and approved by the Research Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology (IORG No: IORG0003571).

Buyang Huanwu Decoction Ameliorates Bleomycin-Induced Pulmonary Fibrosis in Rats via Downregulation of Related Protein and Gene Expression.

Little is known about the effects of Buyang Huanwu decoction on pulmonary fibrosis. Herein, 144 healthy SD rats were randomly divided into six groups: blank control group (B), model control group (M), positive medicine control group (Mp), and high-, moderate-, and low-dose Buyang Huanwu decoction groups (Hd, Md, and Ld). A pulmonary fibrosis model was established by endotracheal injection of bleomycin. On the second day of modeling, the corresponding saline, methylprednisolone suspension, and the three doses of Buyang Huanwu decoction were used to treat the 6 groups of rats by intragastric administration for 7, 14, and 28 consecutive days. After 7, 14, and 28 days of treatment, the mRNA expression of CTGF and AKT, the protein level of CTGF, p-AKT, and collagen types I and III were tested. Finally, we found that the serum collagen type I and III level in Hd, Md, and Ld rats on the 14th and 28th day and the collagen type I and III level in Hd rats on 7th day were significantly lower than in M rats (P < 0.01). The protein level of p-AKT and CTGF in Hd and Md rats on the 7th and 14th days and the protein level of p-AKT in Hd rats on the 28th day were lower than in M rats (P < 0.01, P < 0.05). The level of CTGF mRNA in Hd, Md, and Ld rats and the level of AKT mRNA in Hd and Md rats on the 7th, 14th, and 28th days and the expression level of AKT mRNA in Ld rats on the 14th and 28th days were significantly lower than in M rats (P < 0.01). The study suggests that Buyang Huanwu decoction alleviated pulmonary fibrosis of rats by improvement of lung tissue morphology, low level of serum collagen types I and III, and the reduced expression of CTGF and p-AKT protein, which might be a result of its downregulated expression of CTGF and AKT mRNA levels.

CTGF Is Expressed During Cystic Remodeling in the PKD/Mhm (cy/+) Rat Model for Autosomal-Dominant Polycystic Kidney Disease (ADPKD)

Connective tissue growth factor (CTGF, also named CCN2) plays an important role in the development of tubulointerstitial fibrosis, which most critically determines the progression to end-stage renal failure in autosomal-dominant polycystic kidney disease (ADPKD), the most common genetically caused renal disease. We determined CTGF expression in a well-characterized animal model of human ADPKD, the PKD/Mhm (cy/+) rat. Kidneys of 12 weeks old (cy/+) as well as (+/+) non-affected rats were analyzed for CTGF RNA and protein expression by RT-PCR, Northern and Western blot analyses, in situ hybridization, and IHC. Besides the established expression of CTGF in glomerular cells in kidneys of wild-type (+/+) animals, in (cy/+) rats, CTGF mRNA and protein were robustly expressed in interstitial, stellate-shaped cells, located in a scattered pattern underlying the cystic epithelium and in focal areas of advanced tubulointerstitial remodeling. Renal CTGF mRNA and protein expression levels were significantly higher in (cy/+) rats compared with their (+/+) littermates. Detection of CTGF expression in cells adjacent to cystic epithelium and in areas of marked fibrosis suggests a role in the local response to cyst development and indicates that CTGF may be a relevant factor contributing to tubulointerstitial fibrosis in polycystic kidney disease.