TGF-β1 mRNA Levels Research Articles

Hepatitis B and Hepatitis C Virus Infection Promote Liver Fibrogenesis through a TGF-β1-Induced OCT4/Nanog Pathway.

Hepatitis B virus (HBV)/hepatitis C virus (HCV) coinfection accelerates liver fibrosis progression compared with HBV or HCV monoinfection. Octamer binding transcription factor 4 (OCT4) and Nanog are direct targets of the profibrogenic TGF-β1 signaling cascade. We leveraged a coculture model to monitor the effects of HBV and HCV coinfection on fibrogenesis in both sodium taurocholate cotransporting polypeptide-transfected Huh7.5.1 hepatoma cells and LX2 hepatic stellate cells (HSCs). We used CRISPR-Cas9 to knock out OCT4 and Nanog to evaluate their effects on HBV-, HCV-, or TGF-β1-induced liver fibrogenesis. HBV/HCV coinfection and HBx, HBV preS2, HCV Core, and HCV NS2/3 overexpression increased TGF-β1 mRNA levels in sodium taurocholate cotransporting polypeptide-Huh7.5.1 cells compared with controls. HBV/HCV coinfection further enhanced profibrogenic gene expression relative to HBV or HCV monoinfection. Coculture of HBV and HCV monoinfected or HBV/HCV coinfected hepatocytes with LX2 cells significantly increased profibrotic gene expression and LX2 cell invasion and migration. OCT4 and Nanog guide RNA independently suppressed HBV-, HCV-, HBV/HCV-, and TGF-β1-induced α-SMA, TIMP-1, and Col1A1 expression and reduced Huh7.5.1, LX2, primary hepatocyte, and primary human HSC migratory capacity. OCT4/Nanog protein expression also correlated positively with fibrosis stage in liver biopsies from patients with chronic HBV or HCV infection. In conclusion, HBV and HCV independently and cooperatively promote liver fibrogenesis through a TGF-β1-induced OCT4/Nanog-dependent pathway.

Ferroptosis plays an important role in promoting ionizing radiation-induced intestinal injuries

The intestinal tract is an essential component of the body's immune system, and is extremely sensitive to exposure of ionizing radiation. While ionizing radiation can effectively induce multiple forms of cell death, whether it can also promote ferroptosis in intestinal cells and the possible interrelationship between ferroptosis and intestinal immune function has not been reported so far. Here, we found that radiation-induced major ultrastructural changes in mitochondria of small intestinal epithelial cells and the changes induced in iron content and MDA levels in the small intestine were consistent with that observed during cellular ferroptosis, thus suggesting occurrence of ferroptosis in radiation-induced intestinal damage. Moreover, radiation caused a substantial increase in the expression of ferroptosis-related factors such as LPCAT3 and ALOX15 mRNA, augmented the levels of immune-related factors INF-γ and TGF-β mRNA, and decreased the levels of IL-17 mRNA thereby indicating that ionizing radiation induced ferroptosis and impairment of intestinal immune function. Liproxstatin-1 is a ferroptosis inhibitor that was found to ameliorate radiation-induced ferroptosis and promote the recovery from immune imbalances. These findings supported the role of ferroptosis in radiation-induced intestinal immune injury and provide novel strategies for protection against radiation injury through regulation of the ferroptosis pathway.

Extracellular Vesicles from miR-148a-5p-Enriched Bone Marrow Mesenchymal Stem Cells Relieve Hepatic Fibrosis by Targeting Smad4.

Liver fibrosis is a hallmark feature of many chronic liver diseases, which is the leading cause of morbidity and mortality worldwide. Bone marrow mesenchymal stem cells (BMSCs)-derived extracellular vesicles have been applied in many diseases. In this study, we aimed to explore the specific mechanism of extracellular vesicles from BMSCs in liver fibrosis. Bioinformatics analysis was employed to screen miRNA and its target mRNA. Sirius Red staining was carried out to examine fibrosis in liver tissues. Extracellular vesicle morphology was assessed using Transmission Electron Microscopy. Quantitative real-time PCR (qRT-PCR) and western blotting analysis were performed to detect the expressions of miR-148a-5p, Smad4, transforming growth factor-β1 (TGF-β1), tissue inhibitor of metalloproteinase 1 (TIMP-1), Collagen I, α-smooth muscle actin (α-SMA), and extracellular vesicle markers CD9, TSG101, CD63, and calnexin. Dual-luciferase report gene assay was used for the luciferase activity analysis. Bioinformatics analysis revealed miR-148a-5p as a regulator in liver fibrosis. QRT-PCR results indicated that miR-148a-5p was lowly expressed in both thioacetamide (TAA)-induced mice and TGF-β1-activated hepatic stellate cells. Extracellular vesicles from miR-148a-5p enriched BMSCs downregulated the mRNA and protein levels of TGF-β1, TIMP-1, Collagen I,and α-SMA. Further bioinformatics analysis indicated that Smad4 was related to liver fibrosis. Furthermore, the dual-luciferase report gene assay confirmed the binding relationship between miR-148a-5p and Smad4. Extracellular vesicles from miR-148a-5p enriched BMSCs attenuated hepatic fibrosis in liver fibrosis by targeting Smad4.

Helicobacter hepaticus Infection Promotes the Progression of Liver Preneoplasia in BALB/c Mice via the Activation and Accumulation of High-Mobility Group Box-1

It has been documented that Helicobacter hepaticus (H. hepaticus) infection is linked to chronic hepatitis and fibrosis in male BALB/c mice. However, the mechanism underlying the mice model of H. hepaticus–induced hepatocellular carcinoma is not fully known. In this study, male BALB/c mice were infected with H. hepaticus for 3, 6, 12, and 18 months. H. hepaticus colonization, histopathology, expression of proinflammatory cytokines, key signaling pathways, and protein downstream high-mobility group box-1 (HMGB1) in the liver were examined. Our data suggested that the H. hepaticus colonization level in the colon and liver progressively increased over the duration of the infection. H. hepaticus–induced hepatic inflammation and fibrosis were aggravated during the infection, and hepatic preneoplasia developed in the liver of infected mice at 12 and 18 months post-inoculation (MPI). H. hepaticus infection increased the levels of alanine aminotransferase and aspartate aminotransferase in the infected mice. In addition, the mRNA levels of IL-6, Tnf-α, Tgf-β, and HMGB1 were significantly elevated in the liver of H. hepaticus–infected mice from 3 to 18 MPI as compared to the controls. In addition, Ki67 was increased throughout the duration of the infection. Furthermore, HMGB1 protein was activated and translocated from the nucleus to the cytoplasm in the hepatocytes and activated the proteins of signal transducers and activators of transcription 3 (Stat3) and mitogen-activated protein kinase (MAPK) [extracellular regulated protein kinases 1/2 (Erk1/2) and mitogen-activated protein kinase p38 (p38)] upon H. hepaticus infection. In conclusions, these data demonstrated that male BALB/c mice infected with H. hepaticus are prone to suffering hepatitis and developing into hepatic preneoplasia. To verify the effect of HMGB1 in the progression of liver preneoplasia, mice were infected by H. hepaticus for 2 months before additional HMGB1 recombinant adenovirus treatment. All mice were sacrificed at 4 MPI, and the sera and liver tissues from all of the mice were collected. Immunology and histopathology evaluation showed that HMGB1 knockdown attenuated the H. hepaticus–induced hepatic and fibrosis at 4 MPI. Therefore, we showed that H. hepaticus–induced liver preneoplasia is closely correlated with the activation and accumulation of HMGB1.

Effects of Signaling Activation of Bone Morphogenetic Protein 2 on the Differentiation and Infiltration of Regulatory T Lymphocytes in Tumors

Objective To determine whether the signaling activation of bone morphogenetic protein 2(BMP2)can induce myeloid-derived suppressor cells(MDSC)to secret transforming growth factor β(TGF-β),further enhancing the differentiation and infiltration of regulatory T lymphocytes(Treg)into tumor tissue. Methods The BMP2-induced mRNA and protein expression of TGF-β in MDSC was detected by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay(ELISA),respectively.The effect of BMP2-induced TGF-β secretion by MDSC on Treg differentiation was then determined by flow cytometry.Finally,we implanted the recombined human bone morphogenetic protein 2(rhBMP2)collagen gels into tumor-burdened mice to examine the role of BMP2 in Treg differentiation via MDSC-secreted TGF-β in vivo.The protein levels of TGF-β in peripheral blood and tumor tissue were detected by ELISA,and the infiltration of Treg cells in tumor tissue was detected by immunofluorescence assay. Results BMP2 up-regulated the mRNA and protein levels of TGF-β in MDSC in vitro.TGF-β secreted by the MDSC exposed to BMP2 treatment could induce the differentiation of Treg cells in vitro.Local implantation of rhBMP2 could increase the level of TGF-β protein in peripheral blood and tumor tissue of mice,further enhancing the infiltration of Treg cells into tumors. Conclusion BMP2 signaling activation can induce the differentiation of Treg cells by promoting the secretion of TGF-β in MDSC,and subsequently promote the infiltration of Treg cells into tumors.

Resveratrol attenuates inflammation and apoptosis through alleviating endoplasmic reticulum stress via Akt/mTOR pathway in fungus-induced allergic airways inflammation

BackgroundResveratrol has shown pleiotropic effects against inflammation and oxidative response. The present study aimed to investigate the effects and mechanisms of resveratrol on fungus-induced allergic airway inflammation. MethodsFemale BALB/c mice were injected intraperitoneally with Aspergillus fumigatus (Af) extract emulsified with aluminum on day 0 and 7 and intranasally challenged with Af extracts on day 14 and 15. Resveratrol or dexamethasone or a vehicle was injected intraperitoneally 1 h before each challenge. Mice were sacrificed for serum, bronchoalveolar lavage fluid (BALF), and lungs 24 h after the last challenge. The control group was administered with saline. BEAS-2B was used for the experiments in vitro that Af-exposed airway epithelial cells. ResultsResveratrol and dexamethasone attenuated the airway inflammation and eosinophilia, and reduced not only the production of IL-4, IL-5, and IL-13 in the BALF and lung tissues but also the mRNA levels of lung IL-6, TNF-α, and TGF-β induced by Af challenge (P < 0.05). Furthermore, Af-induced lung endoplasmic reticulum (ER) stress-related proteins PERK, CHOP, and GRP78 and the apoptosis markers including cleaved caspase-3 and cleaved caspase-7 were both suppressed significantly by resveratrol (P < 0.05). In vitro, activation of ER stress and the Akt/mTOR pathway in Af-exposed BEAS-2B cells were effectively ameliorated by resveratrol. Inhibition of the Akt/mTOR pathway using LY294002 suppressed the ER stress while ER stress inhibitor 4-PBA decreased the apoptosis in Af-exposed BEAS-2B cells. ConclusionsOur findings collectively revealed that resveratrol alleviated the Af-exposed allergic inflammation and apoptosis through inhibiting ER stress via Akt/mTOR pathway, exerting therapeutic effects on the fungus-induced allergic lung disorder.

Tranexamic acid inhibits melanogenesis partially via stimulation of TGF-β1 expression in human epidermal keratinocytes.

Oral tranexamic acid (TA) has been an effective treatment for melasma with unclear mechanism. The present study aimed to demonstrate the effect of TA on melanogenesis via regulation of TGF-β1 expression in keratinocytes. We firstly determined the expression level of TGF-β1 in TA-treated keratinocyte-conditioned medium (KCM). Then, the mRNA and protein levels of microphthalmia-associated transcription factor (MITF), tyrosinase (TYR) and tyrosinase-related protein 1 (TRP-1) of human epidermal melanocytes (NHEMs) in the presence of TA-treated KCM were evaluated via RT-PCR and western blot analysis. Moreover, melanin content and tyrosinase activity were quantified. TGF-β1 gene was knocked down by small interfering RNA (siRNA) in keratinocytes. The mRNA and protein levels of TGF-β1 in keratinocytes were significantly increased after TA treatment. Melanin contents, tyrosinase activity, protein and mRNA levels of TYR, MITF and TRP-1 were downregulated in NHEMs in the presence of TA-treated KCM. Knockdown of TGF-β1 in keratinocytes could attenuate the inhibitory effect of TA-treated KCM on melanogenesis. TA could stimulate TGF-β1 expression in keratinocytes, which further inhibits melanogenesis through the paracrine signalling.

Neuroprotective Effects of Hesperetin in Regulating Microglia Polarization after Ischemic Stroke by Inhibiting TLR4/NF-κB Pathway.

This study aimed to explore the influence of hesperidin on the polarization of microglia to clarify the key mechanism of regulating the polarization of M2 microglia. C57BL/6 mice were randomly divided into middle cerebral artery occlusion model group (MCAO group), MCAO + hesperidin treatment group (MCAO + hesperidin group), and sham group (sham operation group). The mice were assessed with neurological scores for their functional status. 2,3,5-Triphenyltetrazole chloride (TTC) was used to determine the volume of cerebral infarction. Hematoxylin and eosin (H&E) staining was performed to detect brain loss. The system with 1% O2, 5% CO2, and 92% N2 was applied to establish BV2 in vitro model induced by MCAO. TNF-α, IL-1β, TGF-β, and IL-10 levels of cytokines in the supernatant were detected by ELISA. RT-qPCR was used to detect mRNA levels of M1 iNOS, CD11b, CD32, and CD86, and mRNA levels of M2 CD206, Arg-1, and TGF-β. The Iba-1, iNOS, and Arg-1 of microglia and protein levels of TLR4 and p-NF-κB related to the pathway were detected by Western blot. After treatment with hesperidin, BV2 cells induced by MCAO in vitro can reduce the proinflammatory cytokines of TNF-α and IL-1β significantly, further upregulating anti-inflammatory cytokines of TGF-β, IL-10 while inhibiting TLR4 and p-NF-κB expression. The MCAO-induced BV2 cells treated by TLR-4 inhibitor TAK-242 and NF-κB inhibitor BAY 11-7082 had similar polarization effects to those treated with hesperidin. This study found that hesperetin gavage treatment can improve the neurological deficit and regulate the polarization of microglia in MCAO mice. In vitro experiments further verified that hesperidin plays a neuroprotective role by inhibiting the TLR4-NF-κB pathway, thus providing new targets and strategies for neuroprotection and nerve repair after ischemic stroke.

Influence of Silica Exposure for Lung Silicosis Rat.

Objective To investigate the influence of silica exposure on the expression of connective tissue growth factor (CTGF), transforming growth factor beta-1 (TGF-β1), and platelet-derived growth factor (PDGF) in lung silicosis rat. Methods Wistar rats were divided into an experimental group and a control group. In the experimental group, rats were exposed to silica by intratracheal instillation. In the control group, rats were exposed to physiological saline by intratracheal instillation. After 45 days, we compared the level of fibrosis and CTGF, TGF-β1, and PDGF in the lungs by immunohistochemistry or reverse transcription-polymerase chain reaction between the two groups. Results The results showed that the expression levels of CTGF, TGF-β1, and PDGF mRNA were significantly higher in the experimental group than those in the control group (P < 0.05). The positive staining of CTGF, TGF-β1, and PDGF mRNA was found in the cytoplasm, especially in the silicotic nodules of the hyalinisation section and cell endochylema of the alveolar macrophages, type II pneumonocytes, and lung tracheal epithelium. There were significantly positive correlations between CTGF, TGF-β1, and PDGF expressions (P < 0.05). A protein–protein interaction analysis showed interactions between TGF-β1, CTGF, and PDGF. Conclusions TGF-β/CTGF signaling pathway plays an important role in silicosis. Silicon dioxide exposure can induce the expression of CTGF, TGF-β1, and PDGF.

Abstract 12855: Left Atrial Reservoir Strain is an Early Marker of Atrial Fibrosis in Cardiovascular Surgery Patients

Introduction: Atrial fibrosis plays an important role in atrial remodeling and the development of atrial fibrillation (AF). Left atrium (LA) strain (LAS) measured by two-dimensional speckle tracking echocardiography (2DS) has been reported to be a useful method to predict the degree of atrial fibrosis. Hypothesis: To investigate whether LAS is an early marker of atrial fibrosis in cardiovascular surgery patients. Methods: Conventional echocardiography and 2DS method were performed in 131 patients (53 AF, 78 sinus rhythm (SR)) including cardiac valve disease (MR, AS, and CABG et al.). The mRNA and microRNA expression were analyzed by real-time reverse transcription polymerase chain reaction (RT-PCR) in 59 cases in which LA tissues were removed from the appendage. Results: Left atrial reservoir strain (LASr) negatively correlated with left atrial volume index (LAVI), and E/e’ and positively correlated with LVEF, and left atrial conduit strain (LAScd). LASr was significantly lower, and LAVI was higher in AF group. The fibrosis-related (COL1A, COL3A1, TGFβ1) and peroxisome proliferator-activated receptor-α (PPARα)-related (PPARα, PGC1α, CPT1B) genes expression as a metabolic remodeling showed a positive association with atrial ANP mRNA expression level, reflecting atrial pressure and mechanical stretching. The expression level of COL1A and TGFβ1 mRNA was higher in AF group, compared with SR group. LASr, but not LAVI, negatively correlated with the expression of COL1A1, TGFβ1, and miR21. Multivariate regression analysis showed that LASr was an independent predictor of COL1A1, TGFβ1, or miR21 expression. The PPARα-related gene expression did not correlate with LASr, and LVEF in SR group, while they had a significant negative correlation with LVEF in AF patients. AF patients (LVEF&lt;58%) had higher expression level of the PPARα-related genes, compared with those (LVEF≥58%). Conclusions: Low LASr exhibits increased atrial fibrosis, and LASr is extremely useful as an early marker for predicting atrial fibrosis and consequently AF in cardiovascular surgery patients. Furthermore, in progressed AF patients, overexpression of PPARα signaling may play a role in atrial degeneration eventually to leading to heart failure.

Effect of fresh Phragmitis Rhizoma on airway inflammation in chronic bronchitis based on TGF-β signaling pathway

This study aims to explore the mechanism of fresh Phragmitis Rhizoma against chronic bronchitis airway inflammation. The SD rats of SPF grade were divided into control group, model group, Guilongkechuanning group(GLKCN, 1.125 g·kg~(-1)), high-dose fresh Phragmitis Rhizoma group(LG-HD, 15 g·kg~(-1)), and low-dose fresh Phragmitis Rhizoma group(LG-LD, 7.5 g·kg~(-1)). The chronic bronchitis models of rats in other groups except the control group were induced by the modified smoking method. From the 15 th day of modeling, the rats were given corresponding agents by gavage for 20 consecutive days. After the last administration, the rats were sacrificed for sample collection. Enzyme-linked immunosorbent assay(ELISA) was employed to detect serum transforming growth factor-β(TGF-β) and interleukin-6(IL-6) levels. The protein expression of TGF-β, IL-1β and IL-6 in lung tissue was detected by immunohistochemical method. Masson staining was performed to detect collagen fibers and muscle fibers in lung tissue, and HE staining to detect the pathological changes of lung tissue. Human bronchial epithelial(16 HBE) cells were cultured in vitro, and CCK-8(cell counting kit-8) method was used to detect the cytotoxicity of cigarette smoke extract(CSE) and fresh Phragmitis Rhizoma. After the exposure of 16 HBE cells to 3.5% CSE and appropriate concentration(800, 400 μg·mL~(-1)) of fresh Phragmitis Rhizoma for 24 h, quantitative real-time PCR was conducted to determine the mRNA levels of TGF-β and IL-1β, and Western blot was employed to determine the protein levels of TGF-β and IL-6 in the cells. The rat model of chronic bronchitis induced by smoking was successfully established. Fresh Phragmitis Rhizoma reduced serum TGF-β and IL-6 levels, down-regulated the protein levels of TGF-β, IL-1β, and IL-6 in lung tissue, and alleviated pathological changes and fibrotic lesions in lung tissue. Moreover, it down-regulated the CSE-induced protein expression of TGF-β and IL-6 as well as the mRNA level of TGF-β in 16 HBE cells. These results indicated that fresh Phragmitis Rhizoma could prevent airway inflammation from chronic bronchitis and promote cell repair by inhibiting the TGF-β signaling pathway.

Effects of Notch-1/Twist-1 axis in the process of epithelial-mesenchymal transition of type II alveolar epithelial cell and its mechanism

Objective: To study the role of Notch-1/Twist-1 axis in the process of epithelial-mesenchymal transition (EMT) of type II alveolar epithelial cells in pulmonary fibrosis (PF) and hope to provide a new theoretical basis for the pathogenesis of PF. Methods: Thirty rats were randomly divided into control group and bleomycin (BLM) group, 15 rats in each group. The PF rat model was induced by intratracheal injection of BLM (7 500 U/kg). Excised inferior lobe of left lung was fixed in 10% formalin for HE staining, Masson staining and transforming growth factor-beta 1 (TGF-β1) immunohistochemistry staining after BLM injection for 28 days. The cultured type II alveolar epithelial cells (RLE-6TN) were divided into 4 groups (Control group, transforming growth factor-beta 1 (TGF-β1) group, Notch-1 negative control siRNA (NC siRNA, 100 pmol/L) group and Notch-1 siRNA (100 pmol/L) group), each group was established nine holes. The cells were treated with TGF-β1 (5.0 ng/ml) for 24 h following NC siRNA or Notch-1 siRNA for 48 h. The mRNA and (or) proteins levels of TGF-β1, collagen I, collagen III, E-Cadherin, zonula occludens-1 (ZO-1), Vimentin, E-Cadherin, Notch-1, Notch-1 intracellular domain (NICD), Hes-1 and Twist-1 were detected in lung tissue and type II alveolar epithelial cells. Results: In vivo, compared with the control group, the alveolar atrophy, collapse and fusion occurred, alveolar septum widened significantly, and a large number of inflammatory cells infiltrated in the pulmonary interstitial of the rats in the BLM group. And compared with control group, BLM obviously increased collagen deposition and collagen I and collagen III expressions, while the expressions of ZO-1 and E-cadherin were decreased, and the expressions of Vimentin and N-cadherin were increased, and concomitantly with increasing Notch-1, NICD, Hes-1 and Twist-1 expression in lung tissues of rats (P＜0.01). In vitro, compared with control group, TGF-β1 treatment obviously induced collagen I, collagen III, Notch-1, NICD, Hes-1 and Twist-1 expressions, and the expressions of E-cadherin and ZO-1 were decreased and the expressions of Vimentin and N-cadherin were increased(P＜0.01). Compared with TGF-β1 group, Notch-1 siRNA treatment significantly inhibited the expressions of Notch-1, NICD, Hes-1 and Twist-1, and the expressions of E-cadherin and ZO-1 were increased and the expressions of Vimentin and N-cadherin were decreased, and also obviously reduced the expressions of collagen I and collagen III induced by TGF-β1 (P＜0.05 or P＜0.01). Conclusion: Notch-1/Twist-1 axis is involved in the EMT process of type II alveolar epithelial cells, suggesting that Notch-1/Twist-1 signaling may be involved in the development of pulmonary fibrosis.

Exploring the Effect of Jiawei Buguzhi Pills on TGF-β-Smad Pathway in Postmenopausal Osteoporosis Based on Integrated Pharmacological Strategy.

Objective To explore the effect of Jiawei Buguzhi Pills (JWBGZP) on the TGF-β-Smad pathway in postmenopausal osteoporosis (PMO) based on integrated pharmacological strategy. Method The ETCM database was used to collect JWBGZP. GeneCards and OMIM databases were utilized to obtain PMO-related genes. Cytoscape was used for network construction and analysis, and DAVID was used for GO and KEGG enrichment analysis of key targets. Animal experiments and cell experiments were conducted to further explore the mechanism. The bone mass density was detected by dual-energy X-ray bone densitometer. The TGF-β1 and Smad4 mRNA in bone tissue were detected by RT-qPCR. The TGF-β1 and Smad4 protein in bone tissue were detected by the western blot. The TGF-β1 and Smad4 protein in osteoblasts were determined by immunohistochemistry. Result A total of 721 JWBGZP potential targets and 385 PMO-related genes were obtained. The enrichment analysis showed that JWBGZP may regulate the TGF-beta signaling pathway, oxidation-reduction process, aging, response to hypoxia, response to ethanol, negative regulation of cell proliferation, PI3K-Akt, HIF-1, and other signaling pathways. The animal experiments showed that compared with the model group, the femoral bone mineral density and lumbar bone mineral density of the JWBGZP group increased (P < 0.05); the expression levels of TGF-β1 and Smad mRNA and proteins in the JWBGZP group were significantly higher (P < 0.05). The cell experiment results showed a large number of osteoblast stained blue-purple and orange-red calcified nodules. The expression levels of TGF-β1 and Smad proteins in the JWBGZP group were significantly higher than those in the blank control group and the sham operation group, and the protein expression levels in the model group were the lowest (P < 0.05). Conclusion JWBGZP may be involved in PI3K-Akt, HIF-1, estrogen, prolactin, and other signaling pathways and regulate MAPK1, AKT1, PIK3CA, JAK2, and other gene targets, regulate bone metabolism, and thereby treat PMO.

Supplementing tributyrin to cottonseed protein concentrate‐based diets can improve growth performance, lipid metabolism and distal intestinal immunity in hybrid grouper ( Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂)

An 8-week trial was performed to evaluate tributyrin effects on growth, disease resistance, lipid metabolizing, distal intestinal inflammation-, tight junctions- and apoptosis-related gene expressions caused by high-level cottonseed protein concentrate (CPC) substitution of fishmeal. Fish were fed one fishmeal (50% fishmeal) and six CPC diets (27.5% fishmeal and 25.2% CPC). Seven isonitrogenous (48.0%) and isolipidic (9.0%) were formulated to contain 0.0 (FM), 0.0 (S00), 0.2 (S02), 0.4 (S04), 0.8 (S08), 1.6 (S16) and 3.2 (S32) g/kg tributyrin. Fish (initial body weight: 7.3 ± 0.0 g) were randomly assigned to 21 300-L fiberglass buckets. Results indicated that optimal tributyrin improved weight gain (WG), specific growth rate (SGR), feed utilization, reduced serum triglyceride and total cholesterol levels. Based on Vibrio alginolyticus challenge test, supplementation with 0.4–3.2 mg/kg tributyrin significantly improved percent survival. Optimal tributyrin enhanced distal intestinal development by increasing fold height/intestinal diameter ratio and goblet cell number, and upregulated jam1, claudin3 and ZO-1; and upregulated TGF-β1, hepcidin, downregulated TNF-α, IL-1β, caspase-2 and caspase-8 mRNA levels. Generally, optimal tributyrin could improve growth performance, disease resistance, hepatic lipid metabolism capacity, distal intestinal immunity and tight junctions. Based on SGR, dietary optimal tributyrin supplementation in hybrid grouper was estimated to be 790.0 mg/kg.

Cardiac Fibrosis Is a Risk Factor for Severe COVID-19.

Increased left ventricular fibrosis has been reported in patients hospitalized with coronavirus disease 2019 (COVID-19). It is unclear whether this fibrosis is a consequence of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection or a risk factor for severe disease progression. We observed increased fibrosis in the left ventricular myocardium of deceased COVID-19 patients, compared with matched controls. We also detected increased mRNA levels of soluble interleukin-1 receptor-like 1 (sIL1-RL1) and transforming growth factor β1 (TGF-β1) in the left ventricular myocardium of deceased COVID-19 patients. Biochemical analysis of blood sampled from patients admitted to the emergency department (ED) with COVID-19 revealed highly elevated levels of TGF-β1 mRNA in these patients compared to controls. Left ventricular strain measured by echocardiography as a marker of pre-existing cardiac fibrosis correlated strongly with blood TGF-β1 mRNA levels and predicted disease severity in COVID-19 patients. In the left ventricular myocardium and lungs of COVID-19 patients, we found increased neuropilin-1 (NRP-1) RNA levels, which correlated strongly with the prevalence of pulmonary SARS-CoV-2 nucleocapsid. Cardiac and pulmonary fibrosis may therefore predispose these patients to increased cellular viral entry in the lung, which may explain the worse clinical outcome observed in our cohort. Our study demonstrates that patients at risk of clinical deterioration can be identified early by echocardiographic strain analysis and quantification of blood TGF-β1 mRNA performed at the time of first medical contact.

3D Transglutaminase Fibronectin Hydrogel Therapy for Healing of Chronic Irradiated Porcine Skin Wounds

PURPOSE/BACKGROUND: Chronic irradiated wounds are characterized by incomplete healing, with no effective therapies reported for the treatment of cutaneous radiation injury. Our group previously demonstrated that fibronectin, a key extracellular matrix glycoprotein involved wound healing, is significantly downregulated in radiation-damaged skin. We further found that an enzymatically crosslinked hydrogel is a suitable construct for sustained and incremental fibronectin release. Our present objective was to investigate the application of this fibronectin hydrogel dressing for the treatment of irradiated wounds in the clinically relevant porcine irradiated wound model. METHODS: We created a chronic irradiation skin injury model in female Yucatan minipigs. Six 1-month-old minipigs underwent irradiation of the right dorsolateral neck region for five consecutive days in 5.5 Gy fractionated doses (total: 27.5 Gy). Following irradiation, the minipigs were allowed 6 weeks of recovery for chronic irradiation skin changes to develop. After recovery, 1 cm × 1 cm full-thickness wounds were created in the irradiated fields. After wound creation, 100 μl of fibronectin hydrogel was topically applied on experimental wounds, and 100 μl of phosphate-buffered saline hydrogel was applied on control wounds. Wound photographs were taken at weekly time intervals to calculate the percentage of wound closure relative to original wound size. Tissues isolated from the wound areas were evaluated histologically for wound healing quality and analyzed for gene and protein levels of radiation injury mediators with quantitative RT-PCR (RT-qPCR) and ELISA. RESULTS: Wounds treated with fibronectin hydrogel demonstrated significantly faster wound closure and decreased scarring than wounds treated with phosphate-buffered saline hydrogel. By postoperative day 21, the mean percentage of wound area relative to original wound size was significantly higher in the control wounds (20.5 ± 2.6%) than in the fibronectin-treated wounds (4.3 + 0.9%). By postoperative day 28, the mean percentage of control wound area was 6.1 ± 2.7% while all fibronectin-treated wounds were fully healed. Picrosirius red staining demonstrated that the fibronectin-treated wounds had decreased total scar area (10.3 ± 2.3 mm2) compared with control wounds (37.7 ± 3.2 mm2). In addition, fibronectin hydrogel treatment was associated with decreased levels of radiation-induced inflammatory mediators, TGF-β1 and SMAD3. RT-qPCR of tissue collected from fibronectin-treated wounds had significantly lower mRNA levels of TGF-β1 (0.40 ± 0.07) compared with levels in control wounds (1.0 ± 0.10). Similarly, RT-qPCR data revealed that relative mRNA levels of SMAD3 were significantly lower in fibronectin-treated wounds (0.34 ± 0.08) than in control wounds (1.0 ± 0.18). Lastly, protein level correlation with ELISA found significantly lower TGF-β1 concentrations in fibronectin-treated wounds (2682 + 515.83 pg/mL) compared with control wounds (5245 + 700.08 pg/mL). CONCLUSIONS: Our novel hydrogel therapy functioned as a moisture-rich dressing and bioactive compound carrier. This dressing addresses irradiated skin fibronectin deficiency with topical glycoprotein supplementation, leading to improved rate and quality of chronic irradiated porcine wound healing.

Comparative study on the cardioprotective effects against ischemia/reperfusion injury of the selective SGLT2 inhibitors, empagliflozin, dapagliflozin and ertugliflozin in non-diabetic mice

Abstract Background/Introduction The sodium glucose co-transporter 2 selective inhibitors (SGLT2i) empagliflozin (EMPA), dapagliflozin (DAPA) and ertugliflozin (ERTU) are established drugs against type 2 diabetes mellitus (T2DM). However, the clinical outcomes among them differ; EMPA and DAPA treatment significantly reduced the frequency of cardiovascular events, while ERTU did not reach this endpoint. Thus, the comparative cardioprotective properties and further mechanistic insights for each SGLT2i should be elucidated. Purpose We have previously proved that chronic EMPA administration reduces infarct size (IS), independently of the presence of T2DM in vivo. We aimed to 1) investigate the comparative cardioprotective effect of EMPA, DAPA and ERTU in terms of IS reduction upon short term administration and 2) reveal the mechanisms of cardioprotection in non-diabetic mice. Methods Adult C57BL/6 mice were randomized into four groups and orally received EMPA (10mg/kg/day), DAPA (9.0mg/kg/day), ERTU (9.7mg/kg/day) or vehicle (5% DMSO in water for injection) for 7 days. The doses of DAPA and ERTU were stoichiometrically equivalent to EMPA's dose that we have previously used as clinically relevant. On the 8th day, mice were subjected to 30' ischemia (I), following by 2h reperfusion (R) and IS was measured. Body weight and fasting blood glucose levels were determined at baseline and at the end of the treatment. Then, we sought to unveil the mechanistic differences among those drugs which are responsible for the observed cardioprotection. Additional mice were randomized into the four groups and were treated for 7 days, as described above. On the 8th day, mice were subjected to 30'I/10'R and the ischemic myocardium was collected. We focused on the cardioprotective signaling of SAFE and RISK pathways and we evaluated the phosphorylation and expression of STAT3, PI3K, Akt and eNOS. Also, we examined the gene expression levels of IL-6 and TGF-β as possible mediators of the SAFE pathway. Results Short term oral administration of EMPA and DAPA reduced myocardial IS (19.6%±2.6 EMPA, 18.0%±3.3 DAPA vs. 35.3%±1.7 Control, p&amp;lt;0.01). On the contrary, administration of ERTU did not reduce IS (30.3%±3.1 ERTU, p=NS). Body weight and glucose levels remained unchanged. Phosphorylation of STAT3(Y705) was increased at the 10th min of R in EMPA and DAPA groups, while was not altered by ERTU. Phosphorylation and expression of PI3K, Akt and eNOS did not differ among groups, indicating that the cardioprotective mechanism is independent of RISK pathway. Real time PCR analysis revealed that il-6 mRNA levels were not affected by the treatments. Notably, we observed increased mRNA levels of tgf-β in both EMPA and DAPA groups, which indicates that cardioprotection is mediated through STAT3/TGF-β crosstalk. Conclusion(s) Short-term EMPA and DAPA, but not ERTU treatment for 7 days reduces IS in healthy, non-diabetic mice through STAT3/TGF-β mediated pathways. Funding Acknowledgement Type of funding sources: None.

Tripterygium wilfordii multiglycoside-induced hepatotoxicity via inflammation and apoptosis in zebrafish.

Tripterygium wilfordii multiglycoside (GTW) is a commonly used compound for the treatment of rheumatoid arthritis (RA) and immune diseases in clinical practice. However, it can induce liver injury and the mechanism of hepatotoxicity is still not clear. This study was designed to investigate GTW-induced hepatotoxicity in zebrafish larvae and explore the mechanism involved. The 72 hpf (hours post fertilization) zebrafish larvae were administered with different concentrations of GTW for three days and their mortality, malformation rate, morphological changes in the liver, transaminase levels, and histopathological changes in the liver of zebrafish larvae were detected. The reverse transcription-polymerase chain reaction (RT-PCR) was used to examine the levels of microRNA-122 (miR-122) and genes related to inflammation, apoptosis, cell proliferation and liver function. The results showed that GTW increased the mortality of zebrafish larvae, while significant malformations and liver damage occurred. The main manifestations were elevated levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), significant liver atrophy, vacuoles in liver tissue, sparse cytoplasm, and unclear hepatocyte contours. RT-PCR results showed that the expression of miR-122 significantly decreased by GTW; the mRNA levels of inflammation-related genes il1β, il6, tnfα, il10, cox2 and ptges significantly increased; the mRNA level of tgfβ significantly decreased; the mRNA levels of apoptosis-related genes, caspase-8 and caspase-9, significantly increased; the mRNA level of bcl2 significantly decreased; the mRNA levels of cell proliferation-related genes, top2α and uhrf1, significantly reduced; the mRNA levels of liver function-related genes, alr and cyp3c1, significantly increased; and the mRNA level of cyp3a65 significantly decreased. In zebrafish, GTW can cause increased inflammation, enhanced apoptosis, decreased cell proliferation, and abnormal expression of liver function-related genes, leading to abnormal liver structure and function and resulting in hepatotoxicity.

Dendrobium Mixture Ameliorates Diabetic Nephropathy in db/db Mice by Regulating the TGF-β1/Smads Signaling Pathway.

Dendrobium mixture (DMix) is an effective treatment for diabetic nephropathy (DN), but the molecular mechanism underlying its action remains unclear. In this study, we investigated whether DMix regulates the transforming growth factor-β1 (TGF-β1)/Smads signal transduction pathway. Twenty-four db/db mice were randomly divided into three groups: the model, DMix, and gliquidone groups, while eight db/m mice were selected as the normal control group. The drug was administered by continuous gavage for 8 weeks. Body weight (BW), kidney weight (KW), kidney index, fasting blood glucose (FBG), blood lipid, 24-hour urinary albumin excretion rate, blood urea nitrogen, and serum creatinine levels were measured. Pathological changes in the renal tissue were observed under a light microscope. Real-time quantitative PCR and immunohistochemical staining were used to detect the mRNA and protein expression levels of TGF-β1 and alpha-smooth muscle actin (α-SMA), respectively, in renal tissues. TGF-β1, Smad2, p-Smad2, Smad3, p-Smad3, and α-SMA expression levels were measured using western blotting. The results showed that DMix significantly reduced the FBG level, BW, KW, and blood lipid level and improved renal function in db/db mice. Histopathology showed that DMix alleviated glomerular mesangial cell proliferation and renal interstitial fibrosis in db/db mice. Additionally, DMix reduced the protein and mRNA expression levels of TGF-β1 and α-SMA and inhibited Smad2 and Smad3 phosphorylation. We conclude that DMix may inhibit renal fibrosis and delay the progression of DN by regulating the TGF-β1/Smads signaling pathway.

Effects of Low- and High-Level Gossypol and Sodium Butyrate Supplementation Under High-Level Gossypol Condition on Growth Performance and Intestinal Health of Hybrid Grouper (Epinephelus fuscoguttatus♀×Epinephelus lanceolatus♂)

The supplementation of gossypol in excess is noted to cause detrimental effects such as the reduction of antioxidant enzymes and disruption of lipid metabolism in animals. Studies regarding the effects of different levels of gossypol are very rare; thus, this study was conducted to evaluate the effects of low and high dietary levels of gossypol and of supplementation with 0.13 % sodium butyrate (NaB) under high gossypol conditions on the growth performance and intestinal health of hybrid grouper (Epinephelus fuscoguttatus♀×Epinephelus lanceolatus♂). Four treatments were used: Feed containing 40% fish meal was used as the control group [fishmeal (FM)], the FM diet plus 0.03% gossypol acetic acid (abbreviated as gossypol) as the low-level gossypol group (gL), FM + 0.15% gossypol was used as the high-level gossypol group (gH), and FM+0.15 % gossypol with 0.13 % NaB as the repair group (gHNaB). All diets were isonitrogenous and isolipidic. The results showed that the gL treatment significantly increased specific growth rate (SGR) and feed utilization; upregulated mRNA levels of distal intestinal transforming growth factor-β1 (tgfβ1), jam, occludin, claudin3, and zo1; and downregulated mRNA levels of il8, ifnγ, and akt. The gH treatment significantly reduced SGR and feed utilization; increased distal intestinal total nitric oxide synthase (NOS) activity and nitric oxide (NO) content; upregulated mRNA levels of distal intestinal tnfα, il1β, il6, ifnγ, caspase2, caspase9, and akt; and downregulated mRNA levels of tgfβ1, jam, and zo1. NaB supplementation significantly increased distal intestinal total NOS activity and NO content; downregulated distal intestinal tnfα, il1β, ifnγ, pi3k p85, and akt mRNA levels; and increased distal intestinal tgfβ1, jam, occludin, and zo1 mRNA levels. Above all, low- and high-level gossypol exhibited positive and negative effects on growth performance, distal intestinal anti-inflammatory capacity, and tight junctions, respectively, in hybrid groupers. NaB supplementation improved distal intestinal anti-inflammatory capacity and tight junctions in hybrid groupers to a certain extent.