TGF-β1 Expression Research Articles

Hydrogen sulfide donor NaHS inhibits formaldehyde-induced epithelial-mesenchymal transition in human lung epithelial cells via activating TGF-β1/Smad2/3 and MAPKs signaling pathways

Formaldehyde (FA) long term exposure leads to abnormal pulmonary function and small airway obstruction of the patients. Hydrogen sulfide (H2S) is one of the recognized gaseous transmitters involved in a wide range of cellular functions. It is unknown the involvement of H2S in FA-induced lung injury. The purpose of this study is to investigate the therapeutic potential and mechanism of H2S on FA-induced epithelial-mesenchymal transition (EMT) of human lung epithelial cells. The cell viability of Beas2B and A549 cells after FA treatment were assessed using MTT assay. The endogenous H2S was visualized by fluorescence microscopy using of the 7-azido-4-methylcoumarin (AzMC). Cell morphology was observed under phase contrast microscope. The mRNAs and proteins level were evaluated by reverse transcription-polymerase chain reaction and western blotting assays. FA treatment downregulated the endogenous H2S levels and the mRNAs and proteins level of H2S synthesizing enzymes, such as cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3-MST) in Beas2B and A549 cells. FA treatment changed the cell morphology of Beas2B cells from cuboid to a spindle-shape, while declined the protein level of E-cadherin and increased the protein level of Vimentin. Moreover, FA treatment increased the proteins level of transforming growth factor-β1 (TGF-β1), phosphorylated-Smad2 (p-Smad2), phosphorylated-Smad3 (p-Smad3), phosphorylated-extracellular signal-regulated kinase (p-ERK), phosphorylated-c-Jun N-terminal kinase (p-JNK), and phosphorylated-P38 (p-P38). Furthermore, the inhibitors of TGF-β receptor type 1 (TGFβRI) and mitogen-activated protein kinases (MAPKs) signaling pathways reversed FA-induced decrease in E-cadherin expression and increase in Vimentin expression in Beas2B cells. Sodium hydrogen sulfide (NaHS) increased the level of H2S, while reversed FA-induced the low expression of E-cadherin and the high expression of Vimentin, TGF-β1, p-Smad2, p-Smad3, p-ERK, p-JNK, and p-P38. These findings indicates FA treatment downregulating the endogenous H2S in human lung epithelial cells. NaHS may inhibit FA-induced EMT in human lung epithelial cells via modulating TGF-β1/Smad2/3 and MAPKs signaling pathways. Therefore, we demonstrated that supplementation of exogenous H2S may inhibit FA-induced lung injury.

Transarterial Embolization Using an Inorganic Phosphate Binder Modulates Immunity and Angiogenesis-related Factors in a Rat Model of Liver Cancer

PurposeTo determine how low inorganic phosphate stress (LIPS) induced by sevelamer transartieral embolization (S-TAE) affects immune regulation and angiogenesis in hepatocellular carcinoma (HCC). Material and MethodsTranscatheter arterial embolization (TAE) using conventional lipiodol plus Poly (vinyl alcohol) (PVA) microsphere and S-TAE were conducted on a McA-RH7777 orthotopic liver tumor model in rats, followed by the assessment of alterations in immunity- and angiogenesis-related factors. The cells were cultured under hypoxic conditions and stimulated with LIPS to analyze the modulation of programmed cell death 1 ligand 1 (PD-L1), vascular endothelial growth factor (VEGFα), and transforming growth factor-β1 (TGF-β1) expression through Western blotting, qRT‒PCR, and immunofluorescence assays. Cell migratory capacity and angiogenesis were also evaluated. ResultsTAE increased the expression of neoplastic PD-L1 and VEGFα, and S-TAE, which depletes intratumoral Pi, downregulated the expression of PD-L1, VEGFα and TGF-β1, and augmented the infiltration of CD8+ T-cells, thereby inhibited angiogenesis and activated anticancer immunity. In vitro, the study demonstrated that LIPS inhibits hypoxia-induced upregulation of PD-L1 expression and the HIF-1α/VEGFα axis. Moreover, LIPS inhibited the tube formation ability of Human Umbilical Vein Endothelial Cells (HUVECs) and the migration ability and epithelial–mesenchymal transition (EMT) process of cancer cells under hypoxic conditions. ConclusionsS-TAE inhibited the expression of PD-L1 and VEGFα, thereby activated anti-tumor immunity and suppressing tumor angiogenesis. All the findings reveal the biology of tumors under low Pi stress and suggest the potential therapeutic value of S-TAE.

Chronic Peroxisome Proliferator-Activated Receptor α/γ and Cannabinoid Receptor 2 Agonist Treatments Attenuated Visceral Adipose Tissue (VAT)–Derived Extracellular Vesicle–Related VAT and Intestinal Abnormalities in Nonalcoholic Steatohepatitis Mice

This study explores the mechanisms and combined effects of chronic peroxisome proliferator-activated receptor (PPAR)α/γ and cannabinoid receptor 2 (CB2R) agonists on visceral adipose tissue (VAT)-derived extracellular vesicle (EVs) release and associated systemic/VAT inflammation, decreased VAT capillary density/fibrosis, and intestinal inflammation/hyperpermeability in nonalcoholic steatohepatitis (NASH) mice. NASH mice receiving 1 month of PPARα/γ agonist aleglitazar (10 mg/kg/day), CB2R agonist JWH015 (3 mg/kg/day) alone or combined. High EV release from VAT of NASH mice was associated with severe systemic/VAT/intestinal inflammation, reduced capillary network of VAT, and intestinal hyperpermeability. Combined JWH015 with aleglitazar treatment significantly suppressed HFD-induced obesity/adiposity, inhibited VAT expansion, reduced VAT inflammation/fibrosis, normalized VAT capillary network, and attenuated intestinal mucosal injury, inflammation, and hyperpermeability in NASH+aleg+JWH015 mice. The inhibition of AT-derived EV release and hypoxia inducible factor (HIF)1α levels in AT-derived EV, normalization of CB2R, PPARα, PPARγ, PPARγ1, PPARγ2, tight junction proteins, VEGF/CD31 expression, and downregulations of HIF1α, MCP-1 and TGFβ1 were observed in the VAT and intestine of the NASH+aleg+jwh015 group. In vitro experiments revealed that PPARα/γ and CB2R activation attenuated NASH AT-derived EV (EVnash)-induced pathogenic changes in the J774/SVEC4-10/Caco2 /3T3-L1 cell system. This study suggested that VAT-derived EVs contribute to the pathogenesis of NAFLD and that combined PPARα/γ and CB2R agonist treatment reduces VAT-released EV release and HIF1/MCP-1 signals to ameliorate hepatic steatosis and VAT/intestine abnormalities of NASH mice.

Potential Oncogenic and Immunosuppressive Roles of Multiple EGF-Like Domains 6 (MEGF6) in Colon Cancer: Insights from Bioinformatic Analysis

Multiple epidermal growth factor-like domains protein 6 (MEGF6) is a high molecular weight protein with several EGF-like domains. Although it has been demonstrated to promote metastasis and chemoresistance in colon cancer cells, its specific oncogenic and immunologic functions remain largely unexplored. This study aims to comprehensively analyze publicly available datasets to define the role of MEGF6 in colon cancer development and immune response modulation. The expression of MEGF6 in colon adenocarcinoma (COAD) and normal tissues at both mRNA and protein levels was assessed using the GEPIA2 and HPA databases, respectively. The UALCAN database was employed to examine the association between MEGF6 expression and clinicopathological outcomes in COAD patients, and survival analysis was conducted using the KM plotter database. Functional enrichment analysis of MEGF6-correlated genes was performed using the ShinyGO online tool. Additionally, the correlation of MEGF6 with immune cell infiltration and immunosuppressive molecules was explored through the TIMER and TISIDB databases. Our analysis revealed significantly higher mRNA and protein expressions of MEGF6 in COAD tissues compared to normal tissues, with associations to cancer stage, nodal metastasis status, and histological subtype. High MEGF6 expression was correlated with poorer survival outcomes, and genes correlated with MEGF6 were enriched in biological processes related to cellular component organization, cell adhesion, and extracellular matrix interaction. Furthermore, MEGF6 expression demonstrated a significant correlation with immune cell infiltration and the expression of immunosuppressive molecules, particularly CTLA-4, PD-1, PD-L1, TGF-β, and IL-10. In conclusion, our findings suggest that MEGF6 may play an oncogenic role by influencing cell and extracellular matrix interactions. Its overexpression may indicate immunosuppressive properties within the cancer microenvironment. Therefore, MEGF6 holds promise as a potential biomarker for predicting both prognosis and therapeutic responses in colon cancer. HIGHLIGHTS MEGF6 has been shown to involve colon cancer growth and chemotherapeutic resistance. Bioinformatics confirmed a prognostic significance of MEGF6 in colon cancer. MEGF6 may exert its oncogenic effects by influencing cell-matrix interactions. MEGF6 expression is correlated with immunosuppressive properties of cancers. GRAPHICAL ABSTRACT

Effects of Shaofu Zhuyu decoction on intestinal flora and fibrosis in a mouse model of endometriosis

Shaofu Zhuyu decoction has been widely used to treat gynecological diseases; however, its mechanism of action in endometriosis remains unclear. We analyzed Shaofu Zhuyu decoction’s chemical composition using ultra-high performance liquid chromatography-mass spectrometry. In an endometriosis mouse model, ectopic lesions weight measurements and hematoxylin and eosin staining were used to assess the therapeutic efficacy of Shaofu Zhuyu decoction. Effects on intestinal microflora were analyzed using 16S ribosomal ribonucleic acid sequencing, and impacts on focal fibrosis were analyzed using Masson’s trichrome staining. Moreover, fibrosis- and metabolism-related proteins were assessed using immunohistochemistry and enzyme-linked immunosorbent assay. The study identified 157 chemical constituents within Shaofu Zhuyu decoction. Shaofu Zhuyu decoction treatment in mice with endometriosis resulted in a reduction in ectopic lesions weight (P<0.05) and delayed disease progression. Moreover, it improved the diversity and abundance of intestinal flora, and decreased the expression of Lachnospiraceae (P<0.05), Rikenellaceae (P<0.01), Ruminococcaceae (P<0.01), Lachnoclostridium (P<0.05), and unclassified_f__Ruminococcaceae (P<0.05). Kyoto Encyclopedia of Genes and Genomes analysis revealed enrichment in carbohydrate, amino acid, and lipid metabolism pathways. Masson’s trichrome staining revealed that compared to the untreated group, the Shaofu Zhuyu decoction group exhibited significantly reduced collagen deposition areas (P<0.001), lower TGF-β1 (P<0.001), COL1A1 (P<0.05), and α-SMA (P<0.01) expression in ectopic lesions, along with increased serum adiponectin (P<0.05), decreased serum leptin (P<0.05), TGF-β1 (P<0.001), and CTGF (P<0.05). Shaofu Zhuyu decoction regulates the intestinal flora of mice with endometriosis while also reducing fibrosis at the lesion site. These findings highlight novel mechanisms for its efficacy in alleviating endometriosis.

Novel injectable polypeptide nanoparticle encapsulated siRNA targeting TGF-β1 and COX-2 for localized fat reduction I: Preclinical invitro and animal models.

Obesity and localized fat accumulation continue to drive the demand for minimally invasive body contouring technologies including injectable compounds for local fat reduction. siRNA offers a potential for an injectable to specifically target and silence genes involved in adipogenesis with minimal inflammatory side effects. This study evaluates the efficacy of STP705, an injectable containing siRNA encapsulated within histidine-lysine polypeptide (HKP) nanoparticles targeting transforming growth factor β1 (TGF-β1) and cyclooxygenase-2 (COX-2), crucial mediators in adipocyte differentiation and fat retention, using invitro, porcine, and murine models. In vitro experiments on mouse preadipocytes and invivo trials using Diet Induced Obese (DIO) mice and Yucatan minipigs were conducted to assess the gene silencing efficiency, tissue localization, pharmacodynamics, and safety profile of STP705. STP705 effectively reduced the expression of TGF-β1 and COX-2, with a notable decrease in adipocyte volume and lipid content without adverse systemic effects. In DIO mice, the HKP-siRNA complex demonstrated precise localization to injected adipose tissue, maintaining significant gene silencing, and detectable levels of siRNA for up to 14 days post-administration. Similar results in minipigs showed a significant reduction in subcutaneous adipose tissue thickness. The results of these studies support the use of targeted siRNA therapy specifically targeting TGF-β1 and COX-2, for localized fat reduction, offering a potential minimally invasive alternative to current fat reduction methods.

ORAI2 is Important for the Development of Early-Stage Postirradiation Fibrosis in Salivary Glands

PurposeAlthough post-irradiation hyposalivation significantly impairs patient quality of life, the underlying mechanisms driving radiation-induced salivary gland fibrosis and hyposalivation remain poorly understood. This study aims to explore the role of calcium-mediated signaling pathways in radiation-induced salivary gland fibrosis. Materials and MethodsPrimary human submandibular gland (SG) cells and C57BL/6J female mouse SGs were exposed to irradiation to model fibrosis development. Following 15 Gy irradiation exposure, RNA sequencing and bioinformatic analysis were conducted on mouse SGs. The effects of Store-Operated Calcium Entry (SOCE) inhibition using SKF96365 and YM58483 on fibrosis markers were assessed in vitro and in vivo. Additionally, the involvement of ORAI2 protein and the newly identified JNK/NFAT1/TGF-β1 signaling axis in SG fibrosis was explored. ResultsWe identified that the calcium release-activated calcium modulator ORAI2 was important in promoting early-stage post-irradiation fibrosis in SGs. Calcium channel signaling was activated in both human patients and irradiated C57BL/6J female mice SGs. Inhibition of SOCE signaling effectively blocked fibrosis in an ORAI2-dependent manner 30 days after irradiation. Our mechanistic studies revealed a novel ORAI2/JNK/NFAT1 axis within the SOCE pathway critical in driving TGF-β1-mediated fibrogenesis. Encouragingly, pharmacological inhibition of NFAT1 significantly mitigated radiation-induced SG fibrosis and restored saliva flow to 84.61% of normal levels in treated mice 30 days after irradiation, without detectable side effects. ConclusionsOur findings highlight the significance of the ORAI2-mediated calcium signaling pathway, specifically via the ORAI2/JNK/NFAT1 axis, in promoting TGF-β1 expression and contributing to the development of early-stage salivary gland fibrosis following irradiation exposure. Targeting the ORAI2/JNK/NFAT1 axis emerges as a promising therapeutic strategy to alleviate radiation-induced hyposalivation and fibrosis, potentially improving the quality of life for patients undergoing radiotherapy.

The effects of extracorporeal shock wave therapy on cutaneous radiation injury in a mouse model.

Although radiation-induced skin injuries are a concern in patients receiving radiation therapy, there are few effective treatments. The aim of this study was to evaluate the protective effects of extracorporeal shock wave therapy (ESWT) on irradiated fibroblasts and mouse skin. In the in vitro study of human dermal fibroblasts, the experimental group was subjected to ESWT following irradiation (20 Gy). The control groups were only irradiated or only subjected to ESWT. At 24 or 48 h post-ESWT, cell viability, cell migration, and mRNA and protein expression were measured. In the in vivo study, the experimental group (7 mice) was treated with ESWT following irradiation (45 Gy). The control group (7 mice) was only irradiated. At 8 weeks post-irradiation, dorsal skin was harvested for histopathologic examination and protein isolation. In dermal fibroblasts, treatment with ESWT increased viability of irradiated cells compared with irradiated-only and untreated cells (p = 0.005). ESWT increased cell migration 24 h post-irradiation (p = 0.002), and decreased TGF-β1 protein expression 48 h post-irradiation (p = 0.024). In mice, ESWT decreased the level of radiation-related skin injury (p = 0.006). Treatment of irradiated skin with ESWT decreased TGF-β1 (p = 0.009) and phospho-Smad3 (p = 0.009) protein expression, as well as decreasing myofibroblasts (p = 0.047) and increasing vessel density (p < 0.001). Our study demonstrated that ESWT alleviated radiation-induced fibrosis by downregulating TGF-β1 expression. It suggests the potential of ESWT for the treatment of radiation-induced fibrosis.

Hydroxychloroquine-Mediated Autophagy Inhibition Shifts Monocyte-Derived Dendritic Cells Toward Immunogenic Phenotype In Vitro

Dendritic cells (DCs) are recognized as the most potent antigen-presenting cells (APCs) and act as intermediaries between the innate and adaptive immune systems. The maturation and function of DCs are significantly influenced by autophagy. Hence, the current research evaluated the effect of hydroxychloroquine (HCQ), an autophagy inhibitor, on the maturation and activation of monocyte-derived DCs. Monocytes were cultured and induced to differentiate into monocyte-derived DCs by the addition of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) cytokines. After five days, the immature DCs were exposed to HCQ and Lipopolysaccharides (LPS), followed by a 24-hour incubation period. On the sixth day, the flow cytometry technique was employed to assess the impact of HCQ on the phenotypic characteristics of the DCs. Finally, the expression of both pro- and anti-inflammatory genes in the HCQ-treated and untreated DCs groups was examined using the real-time PCR method. DCs that were subjected to treatment with HCQ exhibited a notable augmentation in the expression of CD11c, CD86, and HLA-DR, which are markers associated with maturation and antigen presentation. Furthermore, the DCs treated with HCQ demonstrated a significant reduction in the expression of IL-10, TGF-β, and IDO genes, while displaying an increase in the expression of TNF-α and IL-12 when compared to the control group. These findings indicate that targeting autophagy can induce a shift in DCs towards an immunogenic state. However, further investigations are necessary to assess the impact of HCQ-treated DCs on T cell responses both in vitro and in vivo in tumor-bearing animal models.

Gut commensal Alistipes as a potential pathogenic factor in colorectal cancer

Although previous research has shown that inflammation is associated with development of colorectal cancer (CRC), questions remain about whether inflammatory factor-secreting bacteria play a crucial role in CRC development. The potential role of gut microbiota in secreting inflammatory factors involved in the carcinogenesis of CRC among Chinese patients was explored in this study. 16S rRNA sequencing was utilized to evaluate the distinct microbial characteristics between patients with CRC and colorectal adenoma. The serum levels of TNF-α, IL-6 and IL-10 were measured using Enzyme-linked immunosorbent assay (ELISA), while the expression of LRG1 and TGF-β1 in tissues was evaluated by immunohistochemistry. The correlation between gut microbiota and inflammatory factor signaling was analyzed. Compared with the adenoma group, CRC patients exhibit distinct pathologies. Moreover, elevated levels of CEA, erythrocytes and haemoglobin in the blood of CRC patients were found. In addition, CRC patients have significantly higher levels of TNF-α, IL-6, IL-10, LRG1 and TGF-β1. Spearman correlation analysis revealed that LRG1 was positively related to IL-6 and TNF-α, respectively. The correlation analysis results of TGF-β1 were consistent with the above. The abundance of Blautia and Streptococcus was lower in CRC patients, while the relative abundance of Alistipes, Peptostreptococcus and Porphyromonas was significantly elevated. Moreover, positive correlations between Alistipes and inflammatory factor signaling were also found. Our results suggest that gut commensal Alistipes is a key bacterium with pro-inflammatory properties in the CRC carcinogenesis. TNF-α and IL-6 associated with Alistipes might activate LRG1/TGF-β1 signaling which contributed to the carcinogenesis of CRC.

Inactivation of JNK signalling results in polarity loss and cell senescence of Sertoli cell.

As major somatic cells in the testis, Sertoli cell development is precisely regulated by numerous factors, and aberrant development of these cells is associated with male reproductive diseases. JNK signalling is evolutionarily conserved and involved in multiple critical biological processes. Here, we found that the double knockout of Jnk1 and Jnk2 resulted in aberrant localisation of Sertoli cells at early developmental stages, with most Sertoli cells being lost at later stages. Further studies revealed that the inactivation of JNK signalling caused polarity loss in Sertoli cells. In vitro-cultured Jnk1/2-DKO Sertoli cells exhibited a senescence-associated phenotype. Mechanistic studies demonstrate that JNK signalling is likely involved in establishing Sertoli cell polarity by regulating the expression of TGF-β2, mediated by c-Jun. The senescence of Sertoli cells in JNKs-deficient mice is caused by aberrant proteolysis of P27KIP1, mediated by c-Myc. This study demonstrates the role of JNK signalling in Sertoli cell development and functional maintenance, which may also represent an aetiology of male infertility in humans.

Multi-drug loaded electrospinning nanofibers promote healing with less scar formation by remodeling wound microenvironment via modulating TGF-β1/Smads/EN1 signal

Excessive scar formation following severe burn injury, deep trauma or surgical incision is a critical complication and still is a thorny clinical troublesome, which are mainly characterized by the disordered microenvironment during wound healing. A specialty wound dressing remodeling wound microenvironment is urgently desired. Herein, we fabricate a multi-drug loaded nanofiber delivery system for promoting healing and mitigating scar formation by modulating TGF-β1/Smads/EN1 signal, which encapsulates free rhubarb anthraquinones (RAs) and honey in polyvinylpyrrolidone core layer, and borneol in cellulose acetate shell layer by coaxial electrospinning. The nanofibers exhibited a linear morphology with smooth surface, nanometer in diameter, high porosity, ideal water vapor transmission rate and wettability, a biphasic release profile of RAs with the enhanced dissolution under the synergism of honey and borneol. Furthermore, this nanofiber wound dressing possesses the desired biocompatibility and pro-migration effects on HSF cells and HUVEC, long-term effects against Methicillin-resistant Staphylococcus aureus (MRSA) in vitro, remarkably, promoted healing of the MRSA-infected postsurgical wound and severe burn injury characterized by enhanced closure rates and more hair follicles, and mitigated scar formation characterized by less dermal thickness and more organized collagen arrangement by decreasing the expressions of TGF-β1, Smads2/3 and EN1, and increasing Smad7 expression in wound tissues. Collectively, the findings indicate that the multi-drug loaded nanofiber wound dressing can serve as a promising candidate for the application of MRSA-infected wound and severe burn injury healing.

Polysaccharides from Sacha Inchi shell reduces renal fibrosis in mice by modulating the TGF-β1/Smad pathway and intestinal microbiota

Renal fibrosis is a common pathway involved in the progression of various chronic kidney to end-stage diseases, posing a substantial global public health challenge in the search for effective and safe treatments. This study investigated the effects and mechanisms of sacha inchi shell polysaccharide (SISP) on renal fibrosis induced by a high-salt diet (HSD) in mice. By analysing kidney-related protein pathways and the structure of gut microbiota, we found that SISP significantly reduced urinary protein levels induced by a HSD from 41.08 to 22.95 μg/mL and increased urinary creatinine from 787.43 to 1294.50 μmol/L. It reduced renal interstitial collagen fibres by 11.30 %, thereby improving the kidney function. SISP lowered the mRNA expression of TGF-B1, fibronectin, α-SMA, Smad2/3, and TGFBRII, leading to decreased protein levels of TGF-β1, p-Smad2/3, p-TGFβRII, fibronectin, α-SMA, p-Smad2/3/Smad2/3, and p-TGFβRII/TGFβRII. These changes blocked downstream transcription in the TGF-β1/Smad signalling pathway, thereby attenuating renal fibrosis in HSD mice. In addition, SISP altered the intestinal flora imbalance in HSD mice by reducing the relative abundance of the genera, Akkermansia, Faecalibaculum, and unidentified_Ruminococcaceae, and reversing the decline in the levels of the genera, Lactobacillus and Bacteroides. In conclusion, SISP is a promising nutraceutical for renal fibrosis management.

Hepatoprotective action mechanism and quantification of soyasaponin Bb in Abri Herba by HPLC and network pharmacology

Ethnopharmacological relevanceThe herb of Abrus cantoniensis Hance (AC) is an important Traditional Chinese Medicine (TCM) and is also used as an herbal tea with hepatoprotective action. Soyasaponin Bb is one of the pharmacodynamic substances of AC for the herb's effective pharmacological activity. This study aims to investigate the anti-fibrotic and hepatoprotective activities of soyasaponin Bb in vivo and in vitro experiments, mechanism by network pharmacology and quantification by HPLC. Materials and methodsHigh-performance liquid chromatography (HPLC) was applied to evaluate the quality of the herb and determine the contents of soyasaponin Bb from different sources and parts of the AC. In vivo experiments were conducted to induce an acute liver injury model by injecting CCl4 into mice, and an in vitro hepatic fibrosis model was established by cultivating LX-2 cells with TGF-β1. These models were used to explore the anti-fibrotic and hepatoprotective effects of soyasaponin Bb and its underlying mechanisms. In addition, the potential target genes corresponding to soyasaponin Bb were identified using the Swiss Target Prediction database through network pharmacology methods. Meanwhile, hepatic fibrosis targets were screened using the GeneCards, TTD, and OMIM disease databases. The STING database was used to construct the protein-protein interaction (PPI) network of soyasaponin Bb-hepatic fibrosis. The soyasaponin Bb-hepatic fibrosis disease target-pathway network was constructed using Cytoscape 3.9.1 software. Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to enrich and analyze the common targets of the drug and the disease, aiming to identify the potential targets and pathways involved in the anti-fibrotic and hepatoprotective effects of soyasaponin Bb. ResultsThe content of soyasaponin Bb varied across different sources, with the roots containing the highest concentration, up to 0.2480%. In vivo experiments showed that soyasaponin Bb had a protective effect against CCl4-induced acute liver injury, effectively inhibiting the increase in ALT and AST levels and slowing down the hepatocyte inflammatory damage caused by CCl4. Soyasaponin Bb also down-regulated MDA levels and up-regulated SOD levels, indicating a certain antioxidant capacity. In vitro cell experiments showed that soyasaponin Bb could effectively inhibit the proliferation of HSC-LX2 cells induced by TGF-β1 by regulating the TGF-β1/α-SMA pathway, significantly down-regulate the protein expression of TGF-β1 and α-SMA, while also reducing the levels of inflammatory cytokines IL-6 and IL-1β. Further network pharmacology analysis suggested that soyasaponin Bb can exert anti-fibrosis activity by regulating the IBD signaling pathway, Th17 signaling pathway, Hepatitis B signaling pathway, and JAK-STAT signaling pathway. ConclusionSoyasaponin Bb is primarily distributed in the root of AC, and it has a strong protective effect against CCl4-induced acute liver injury. It can reduce the level of inflammatory factors, relieve inflammation, and exert anti-fibrotic activity by regulating the TGF-β1/α-SMA pathway. Its potential anti-hepatic fibrosis mechanism has been investigated through network pharmacology.

Effect of Dietary Copper on Growth Performance, Antioxidant Capacity, and Immunity in Juvenile Largemouth Bass (Micropterus salmoides)

This study evaluated the optimal dietary copper (Cu) levels and their effects on growth performance, body composition, and antioxidant capacity in juvenile largemouth bass (Micropterus salmoides). A total of 360 fish (initial average weight (1.67 ± 0.01 g) and initial average length (2.5 ± 0.2 cm)) were randomly assigned to 18 tanks, each containing 20 fish and six dietary Cu concentrations: 2.13 (control), 3.00, 3.66, 4.58, 4.64, and 5.72 mg/kg. The results indicated that fish receiving 3.00 mg/kg of Cu exhibited the best final body weight (FBW), weight gain rate (WGR), and specific growth rate (SGR), with a significantly reduced feed conversion ratio (FCR). While body composition (moisture, protein, lipid, and ash) remained consistent across groups, plasma total protein (TP) levels increased with Cu supplementation. Elevated triglycerides (TG) and albumin (ALB) were noted at 4.64 and 5.72 mg/kg, respectively, while glucose (GLU) levels decreased with an increase in dietary Cu. Antioxidant capacity, assessed via hepatic glutathione (GSH) and the activities of catalase (CAT), and showed significant improvements at 3.00 and 3.66 mg/kg Cu, while superoxide dismutase (SOD) showed the highest activity at a dietary Cu level of 5.72 mg/kg. Additionally, the expressions of tgf-β and tnf-α genes were significantly upregulated at a dietary Cu level of 5.72 mg/kg, while il-8 and il-10 genes were upregulated at dietary 3.66 mg/kg. The expression of nrf2 was significantly upregulated in response to a dietary Cu level of 3.66 mg/kg compared to the control group, and the expression of the keap1 gene was significantly upregulated in the fish fed with 5.72 mg/kg of dietary Cu. The results indicated that appropriate dietary supplementation could promote the growth performance and improve the antioxidant status the immunity of largemouth bass, and the optimal Cu requirement for FCR and SGR were approximately 3.10 mg/kg and 3.00 mg/kg, respectively.

The mechanism of TGF-β mediating BRD4/STAT3 signaling pathway to promote fibroblast proliferation and thus promote keloid progression

ObjectiveThe purpose of this study was to investigate the effect of TGF-β on keloid and its molecular mechanism in fibroblasts. Methods: The difference between normal tissue and keloid tissue can be detected using HE staining. Fibroblasts were treated with TGF-β, and then treated with the BRD4 inhibitor JQ1 and the STAT3 activator Colivelin TFA. Western blot was used to measure the relative protein expression of TGF-β, BRD4, p-STAT3, p-EZH2, C-myc, KLF2, KLF4, α-SMA, and Collagen-I. Immunofluorescence staining was used to measure the relative fluorescence intensity of BRD4, p-STAT3, α-SMA, and Collagen-I. Cell proliferation ability was evaluated by CCK-8 assay and colony formation assay. Results:The expression of TGF-β and BRD4 was significantly higher in keloid tissue compared to normal tissue. TGF-β mediated the BRD4/STAT3 signaling pathway to inhibit p-EZH2 and promote the expression of C-myc, KLF2, KLF4, α-SMA, and Collagen-I. Additionally, TGF-β mediated the BRD4/STAT3 signaling pathway to enhance fibroblast proliferation. Conclusion:TGF-β mediates the BRD4/STAT3 signaling pathway to promote fibroblast proliferation and contribute to the progression of keloid.

Effect of Ritanserin and Duloxetine on the Gene Expression of Primary Aniridia and Healthy Human Limbal Stromal Cells, In Vitro.

In congenital aniridia caused by mutations in paired box 6 (PAX6), PAX6 influences the migration and differentiation of limbal epithelial cells (LECs), thereby playing a pivotal role in aniridia-associated keratopathy. The antidepressants ritanserin and duloxetine affect PAX6 expression in LECs. Limbal stromal cells, which support limbal epithelial stem cells, are crucial in the limbal stem cell niche. This study explores how ritanserin and duloxetine influence gene expression in primary human limbal stromal cells from subjects with congenital aniridia and from healthy subjects, in vitro. Primary human limbal stromal cells from corneas affected by aniridia (AN-LSCs) (n = 8) and from healthy corneas (LSCs) (n = 8) were isolated and cultured in either low-glucose serum-free (LGSF) or normal-glucose serum-containing (NGSC) media. Cells were treated with 4µM ritanserin or duloxetine for 24h. Quantitative PCR (qPCR) and western blot were used to assess the expression of PAX6, FOSL2, TGF-β1, ACTA2A1, LUM, COL1A1, COL5A1, DSG1, FABP5 and ADH7. In AN-LSCs with LGSF-medium, ritanserin increased PAX6 messenger RNA (mRNA) (p = 0.007) and decreased TGF-β1 and FOSL2 mRNA levels (P = 0.005, P = 0.038). In addition, TGF-β1 protein levels decreased with both treatments (P = 0.02, P = 0.007), and FABP5 protein level increased, using ritanserin (P = 0.019). In LSCs with LGSF-medium, ACTA2A1 mRNA levels decreased using ritanserin and duloxetine (P = 0.028; P = 0.031), while FABP5 mRNA levels increased with ritanserin treatment (P = 0.003). Also, duloxetine use reduced α-SMA protein (P = 0.013) and increased FABP5 protein levels (P = 0.029). In LSCs with NGSC-medium, ritanserin elevated LUM, FABP5 and ADH7 mRNA and protein levels (P = 0.025, P = 0.003, P = 0.047, P = 0.024, P = 0.013, P = 0.039). The results of our study confirmed that the antipsychotropic drugs ritanserin and duloxetine alter PAX6 and TGF-β1 gene expression in AN-LSCs cultured in LGSF-medium. These drugs were found to have an impact on retinoic acid signaling pathways and keratocyte characteristic markers both in LSCs and AN-LSCs, using different culture media.

Gastrodin improves microglia-mediated inflammatory response after hypoxic-ischemic brain damage in neonatal rats via PI3K/AKT pathway

To investigate the mechanism of gastrodin for inhibiting microglia-mediated inflammation after hypoxicischemic brain damage (HIBD) in neonatal rats. Thirty-nine 3-day-old SD rats were randomly divided into sham group, HIBD group and gastrodin treatment group. Western blotting was used to detect the expressions of TNF-α, IL-1β, IL-10 and TGF- β1 in the corpus callosum of the rats. The potential targets of gastrodin for treatment of HIBD were screened by network pharmacology analysis. The expressions of PI3K/AKT signaling pathway proteins following HIBD-induced microglial activation in the rats and in cultured microglial BV-2 cells with oxygen-glucose deprivation (OGD) were detected with Western blotting. The effects of LY294002 (a specific inhibitor of the PI3K/AKT pathway) and gastrodin on TNF-α and TGF-β1 mRNA levels in BV-2 cells with OGD was detected with RT-qPCR. In the neonatal rats with HIBD, gastrodin treatment significantly decreased TNF-α and IL-1β expressions and enhanced IL-10 and TGF-β1 expressions in the ischemic corpus callosum. Network pharmacology analysis showed significant enrichment of the PI3K/AKT signaling pathway and a strong binding between gastrodin and PI3K. Gastrodin significantly promoted PI3K and AKT phosphorylation in neonatal rats with HIBD and in BV-2 cells exposed to OGD. In BV-2 cells with OGD, gastrodin obviously suppressed OGD-induced increase of TNF-α and reduction of TGF-β1 mRNA expressions, and this effect was strongly attenuated by LY294002 treatment. Gastrodin can inhibit microglia-mediated inflammation in neonatal rats with HIBD by regulating the PI3K/AKT signaling pathway.

High temperature induces oxidative stress in spotted seabass (Lateolabrax maculatus) and leads to inflammation and apoptosis

Our study aims to examine the changes of long-term high temperature on the mortality and health status of spotted seabass (Lateolabrax maculatus), as well as to screen suitable biomarkers to determine whether the spotted seabass is under heat stress. In this study, 360 juvenile spotted seabass were evenly distributed into three temperature-controlled systems at 27 °C (N, normal temperature), 31 °C (M, moderate temperature), and 35 °C (H, high temperature) for an 8-week aquaculture experiment. The results revealed that 35 °C water temperature significantly increased the mortality and the MDA content in tissues (P < 0.05). Meanwhile, 35 °C water temperature significantly increased the activity of SOD enzyme and T-AOC capacity in tissues, as well as the expression of hsp60, hsp70, and hsp90 (P < 0.05). Additionally, the expression of nrf2, il1β, il8, caspase3, caspase9, and bax in the liver significantly increased (P < 0.05), while the expression of keap1, il10, tgfβ, and bcl2 decreased significantly (P < 0.05). These results indicate that 35 °C water temperature induces oxidative stress in spotted seabass, leading to tissue oxidative damage, promoting inflammation and apoptosis in liver, and increasing mortality. However, the organism compensates by heightening its antioxidant capacity via the Nrf2-Keap1 signaling pathway and inducing high expression of heat shock proteins for self-protection. Furthermore, the alterations in the mRNA level of hsp70 and MDA content in the liver, muscle, and kidney can serve as indicators for evaluating spotted seabass under prolonged heat stress.

Long non-coding RNA PVT1 regulates TGF-β and promotes the proliferation, migration and invasion of hypopharyngeal carcinoma FaDu cells

Hypopharyngeal carcinoma is one of the malignant tumors of the head and neck with a particularly poor prognosis. Recurrence and metastasis are important reasons for poor prognosis of hypopharyngeal cancer patients, and malignant proliferation, migration, and invasion of tumor cells are important factors for recurrence and metastasis of hypopharyngeal cancer. Therefore, elucidating hypopharyngeal cancer cells’ proliferation, migration, and invasion mechanism is essential for improving diagnosis, treatment, and prognosis. Plasmacytoma Variant Translocation 1 (PVT1) is considered a potential diagnostic marker and therapeutic target for tumors. However, it remains unclear whether PVT1 is related to the occurrence and development of hypopharyngeal cancer and its specific mechanism. In this study, the promoting effect of PVT1 on the proliferation, migration, and invasion of hypopharyngeal carcinoma FaDu cells was verified by cell biology experiments and animal studies, and it was found that PVT1 inhibited the expression of TGF-β, suggesting that PVT1 may regulate the occurrence and development of hypopharyngeal carcinoma FaDu cells through TGF-β.