TL1A Promotes Fibrogenesis in Colonic Fibroblasts via the TGF-ß1/Smad3 Signaling Pathway.

OBJECTIVE: Intestinal fibrosis is a refractory complication of inflammatory bowel disease (IBD). Tumor necrosis factor ligand-related molecule-1A (TL1A) is important for IBD-related intestinal fibrosis in a dextran sodium sulfate (DSS)-induced experimental colitis model. This study aimed to explore the effects of TL1A on human colonic fibroblasts. METHODS: A trinitrobenzene sulfonic acid (TNBS)-induced experimental colitis model of LCK-CD2-TL1A-GFP transgenic (Tg) or wild-type (WT) mice was established to determine the effect and mechanism of TL1A on intestinal fibrosis. The human colonic fibroblast CCD-18Co cell line was treated concurrently with TL1A and human peripheral blood mononuclear cell (PBMC) supernatant. The proliferation and activation of CCD-18Co cells were detected by BrdU assays, flow cytometry, immunocytochemistry and Western blotting. Collagen metabolism was tested by Western blotting and real-time quantitative polymerase chain reaction (RT-qPCR). RESULTS: The level of collagen metabolism in the TNBS+ethyl alcohol (EtOH)/Tg group was greater than that in the TNBS+EtOH/WT group. Transforming growth factor-ß1 (TGF-ß1) and p-Smad3 in the TNBS+EtOH/Tg group were upregulated as compared with those in the TNBS+EtOH/WT group. The proliferation of CCD-18Co cells was promoted by the addition of human PBMC supernatant supplemented with 20 ng/mL TL1A, and the addition of human PBMC supernatant and TL1A increased CCD-18Co proliferation by 24.4% at 24 h. TL1A promoted cell activation and increased the levels of COL1A2, COL3A1, and TIMP-1 in CCD-18Co cells. Treatment of CCD-18Co cells with TL1A increased the expression of TGF-ß1 and p-Smad3. CONCLUSION: TL1A promotes TGF-ß1-mediated intestinal fibroblast activation, proliferation, and collagen deposition and is likely related to an increase in the TGF-ß1/Smad3 signaling pathway.

Tumor Infiltrating Effector Regulatory T Cells Express VEGF Receptor 2 in Patients With Colorectal Cancer.

BACKGROUND/AIM: Regulatory T cells (Tregs) suppress various anti-tumor immune responses in the tumor microenvironment (TME) and their control is considered essential to enhancing efficacy of cancer immunotherapy. The purpose of the study was to evaluate the strategy to regulate Tregs through the vascular endothelial growth factor (VEGF) pathway. MATERIALS AND METHODS: We evaluated VEGF receptor (VEGFR) expression in subtypes of Tregs by analysis of public databases and through flow cytometry by investigating surgically resected specimens and peripheral blood mononuclear cells (PBMCs) from 26 patients with advanced colorectal cancer (CRC). RESULTS: Analysis of The Cancer Genome Atlas colorectal adenocarcinoma dataset (n=592) showed that mRNA expression of both FLT1 (VEGFR1) and KDR (VEGFR2) was positively correlated with mRNA expression of FOXP3 as well as Treg signature. Clinical specimens revealed abundant VEGFR2 expression on Tregs, but very marginal VEGFR1 expression. The frequency of effector Tregs, the most immunosuppressive fraction of Tregs, was significantly higher in the tumor than in the PBMC and normal mucosa, and the majority of effector Tregs expressed VEGFR2. Furthermore, by using in vitro generated Tregs, the proportion of Tregs expressing IL-10 or TGF-ß1 was significantly inhibited by a VEGFR2 inhibitor. CONCLUSION: A therapeutic strategy targeting the VEGFR2 axis may have a potential to control effector Tregs in the CRC-TME.

Membrane Formed Around Liquid Nitrogen-treated Bone Promotes Osteogenesis and Revitalization in a Rat Model.

BACKGROUND/AIM: This study aimed to investigate the structure and functions of the membrane formed around liquid nitrogen-treated bones in the osteogenesis and revitalization of frozen bone using a rat model. MATERIALS AND METHODS: Segmental defects were created in femurs of rats, and resected bones treated with liquid nitrogen [frozen bone (FB) group, n=20] or polymethylmethacrylate (PMMA group; n=20) were implanted as spacers. Histological analysis and quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) of the membrane around each spacer were performed for bone morphogenetic protein 2 (BMP2), transforming growth factor (TGF)-ß1, and vascular endothelial growth factor (VEGF). Furthermore, in week 2, spacers were removed from both groups (n=5 each), and autologous cancellous bone (ACB) harvested from the ilium was grafted into the defect. Radiological analysis was performed until bone union was observed. RESULTS: In week 2, similar two-layered membrane structures were observed in both groups; these matured into fibrous tissues over time. At each evaluation point, qRT-PCR showed higher expression of all factors in the FB than in the PMMA group. In the ACB graft model, the mean period to bone union and new bone volume were significantly shorter and greater, respectively, in the FB. Chondrocytes invaded the osteotomy site from the membrane in the FB, suggesting that endochondral ossification may occur and be related to osteogenesis. Additionally, fibroblasts and capillaries in the membrane invaded the surface of treated bone in week 2, and osteocytes were observed around them in weeks 6 and 8. CONCLUSION: Fibrous membranous tissue formed around liquid nitrogen-treated bones may be vital for osteogenesis and revitalization of frozen bones.

Polystyrene nanoplastics induce cardiotoxicity by upregulating HIPK2 and activating the P53 and TGF-ß1/Smad3 pathways.

Nanoplastics (NPs) pollution has become a global environmental problem, raising numerous health concerns. However, the cardiotoxicity of NPs exposure and the underlying mechanisms have been understudied to date. To address this issue, we comprehensively evaluated the cardiotoxicity of polystyrene nanoplastics (PS-NPs) in both healthy and pathological states. Briefly, mice were orally exposed to four different concentrations (0 mg/day, 0.1 mg/day, 0.5 mg/day, and 2.5 mg/day) of 100-nm PS-NPs for 6 weeks to assess their cardiotoxicity in a healthy state. Considering that individuals with underlying health conditions are more vulnerable to the adverse effects of pollution, we further investigated the cardiotoxic effects of PS-NPs on pathological states induced by isoprenaline. Results showed that PS-NPs induced cardiomyocyte apoptosis, cardiac fibrosis, and myocardial dysfunction in healthy mice and exacerbated cardiac remodeling in pathological states. RNA sequencing revealed that PS-NPs significantly upregulated homeodomain interacting protein kinase 2 (HIPK2) in the heart and activated the P53 and TGF-beta signaling pathways. Pharmacological inhibition of HIPK2 reduced P53 phosphorylation and inhibited the activation of the TGF-ß1/Smad3 pathway, which in turn decreased PS-NPs-induced cardiotoxicity. This study elucidated the potential mechanisms underlying PS-NPs-induced cardiotoxicity and underscored the importance of evaluating nanoplastics safety, particularly for individuals with pre-existing heart conditions.

Time-Dependent Sequential Changes of IL-10 and TGF-ß1 in Mice with Deep Vein Thrombosis.

OBJECTIVES: To detect the expression changes of interleukin-10 (IL-10) and transforming growth factor-ß1 (TGF-ß1) during the development of deep vein thrombosis in mice, and to explore the application value of them in thrombus age estimation. METHODS: The mice in the experimental group were subjected to ligation of inferior vena cava. The mice were sacrificed by excessive anesthesia at 1 d, 3 d, 5 d, 7 d, 10 d, 14 d and 21 d after ligation, respectively. The inferior vena cava segment with thrombosis was extracted below the ligation point. The mice in the control group were not ligated, and the inferior vena cava segment at the same position as the experimental group was extracted. The expression changes of IL-10 and TGF-ß1 were detected by immunohistochemistry (IHC), Western blotting and real-time qPCR. RESULTS: IHC results revealed that IL-10 was mainly expressed in monocytes in thrombosis and TGF-ß1 was mainly expressed in monocytes and fibroblast-like cells in thrombosis. Western blotting and real-time qPCR showed that the relative expression levels of IL-10 and TGF-ß1 in each experimental group were higher than those in the control group. The mRNA and protein levels of IL-10 reached the peak at 7 d and 10 d after ligation, respectively. The mRNA expression level at 7 d after ligation was 4.72±0.15 times that of the control group, and the protein expression level at 10 d after ligation was 7.15±0.28 times that of the control group. The mRNA and protein levels of TGF-ß1 reached the peak at 10 d and 14 d after ligation, respectively. The mRNA expression level at 10 d after ligation was 2.58±0.14 times that of the control group, and the protein expression level at 14 d after ligation was 4.34±0.19 times that of the control group. CONCLUSIONS: The expressions of IL-10 and TGF-ß1 during the evolution of deep vein thrombosis present time-dependent sequential changes, and the expression levels of IL-10 and TGF-ß1 can provide a reference basis for thrombus age estimation.

Targeted Antisense Oligonucleotide-Mediated Skipping of Murine Postn Exon 17 Partially Addresses Fibrosis in D2.mdx Mice.

Periostin, a multifunctional 90 kDa protein, plays a pivotal role in the pathogenesis of fibrosis across various tissues, including skeletal muscle. It operates within the transforming growth factor beta 1 (Tgf-ß1) signalling pathway and is upregulated in fibrotic tissue. Alternative splicing of Periostin's C-terminal region leads to six protein-coding isoforms. This study aimed to elucidate the contribution of the isoforms containing the amino acids encoded by exon 17 (e17+ Periostin) to skeletal muscle fibrosis and investigate the therapeutic potential of manipulating exon 17 splicing. We identified distinct structural differences between e17+ Periostin isoforms, affecting their interaction with key fibrotic proteins, including Tgf-ß1 and integrin alpha V. In vitro mouse fibroblast experimentation confirmed the TGF-ß1-induced upregulation of e17+ Periostin mRNA, mitigated by an antisense approach that induces the skipping of exon 17 of the Postn gene. Subsequent in vivo studies in the D2.mdx mouse model of Duchenne muscular dystrophy (DMD) demonstrated that our antisense treatment effectively reduced e17+ Periostin mRNA expression, which coincided with reduced full-length Periostin protein expression and collagen accumulation. The grip strength of the treated mice was rescued to the wild-type level. These results suggest a pivotal role of e17+ Periostin isoforms in the fibrotic pathology of skeletal muscle and highlight the potential of targeted exon skipping strategies as a promising therapeutic approach for mitigating fibrosis-associated complications.

Semaglutide May Ameliorate Fibrosis and Inhibit Epithelial-Mesenchymal Transition in Intrauterine Adhesion Models.

The purpose of this study was to explore the effect of Semaglutide on intrauterine adhesions and discover new drugs for such adhesions. In this study, the cell model was simulated by TGF-ß1-induced human endometrial epithelial cells, and the animal model was established through mechanical curettage and inflammatory stimulation. After co-culturing with TGF-ß1 with or without different concentrations of Semaglutide for 48 h, cells were collected for RT-qPCR and Western blotting analyses. Three doses were subcutaneously injected into experimental mice once a day for two weeks, while the control group received sterile ddH2O. The serum and uterine tissues of the mice were collected. HE and Masson staining were used for the uterine histomorphological and pathological analyses. RT-qPCR and Western blotting were used for mRNA and protein expression analyses. Serum indicators were detected using ELISA kits. The results showed that Semaglutide significantly reduced the mRNA levels of fibrosis indicators ACTA2, COL1A1, and FN and inflammatory indicators TNF-α, IL-6, and NF-κB in the two models. Semaglutide improved endometrium morphology, increased the number of endometrial glands, and reduced collagen deposition in IUA mice. The results also showed that Semaglutide could inhibit vimentin, E-Cadherin, and N-Cadherin in the two models. In summary, Semaglutide can ameliorate fibrosis and inflammation of intrauterine adhesions as well as inhibit epithelial-mesenchymal transition in IUA models.

Significant Association of Candidate Genes (AGTR1 and TGF-Β1) Polymorphism with Diabetic Nephropathy in Diabetes Mellitus Type 2 Patients.

BACKGROUND/AIMS: Diabetic nephropathy (DN) is one of the complications of diabetes mellitus (DM). This study aimed to investigate the association between genetic polymorphisms, specifically AGTR1 (rs5186) and TGF-ß1 (rs1800470), and the risk of developing Diabetic nephropathy (DN) in type 2 diabetes mellitus patients, compared to those without DN and healthy controls. METHODS: A case-control study was conducted on 165 diabetic patients (59 with diabetic nephropathy (DN) and 54 without DN (DM)), and 52 healthy controls (HC). The genotyping was done using amplification refractory mutation system method (ARMS-PCR). Age, gender, and duration of diabetes were matched across groups. Clinical parameters including FBS, RBS, HbA1C, creatinine, urea, SBP, DBP, total cholesterol, triglycerides, LDL, and BMI were assessed. RESULTS: Diabetic patients with nephropathy exhibited significantly higher levels of clinical parameters compared to those without nephropathy and healthy controls. The risk allele of AGTR1 , C (p <0.0001), and risk allele containing genotypes AC (p <0.0001) and CC (p - 0.0010) were significantly higher in DN patients compared to DM and HC groups. Similarly, the TGF-ß1 risk allele C (p - 0.0001), and corresponding genotypes TC (p - 0.0038) and CC (p - 0.0027) were significantly associated with increased risk of diabetic nephropathy compared to DM and HC groups. CONCLUSION: The data showed significant association of AGTR1 (rs5186) and TGF-ß1 (rs1800470) polymorphism with an increased risk of diabetic nephropathy in type 2 diabetes mellitus patients. More investigation will be required to disseminate the results, while increasing the samples size and using whole genome sequencing.

TRPC6 Knockout Alleviates Renal Fibrosis through PI3K/AKT/GSK3B Pathway.

OBJECTIVE: Renal fibrosis is the ultimate pathway of various forms of acute and chronic kidney damage. Notably, the knockout of transient receptor potential channel 6 (TRPC6) has shown promise in alleviating renal fibrosis. However, the regulatory impact of TRPC6 on renal fibrosis remains unclear. METHODS: In vivo, TRPC6 knockout (TRPC6-/-) mice and age-matched 129 SvEv (WT) mice underwent unilateral renal ischemia-reperfusion (uIR) injury surgery on the left renal pedicle or sham operation. Kidneys and serum were collected on days 7, 14, 21, and 28 after euthanasia. In vitro, primary tubular epithelial cells (PTECs) were isolated from TRPC6-/- and WT mice, followed by treatment with transforming growth factor ß1 (TGFß1) for 72 h. The anti-fibrotic effect of TRPC6-/- and the underlying mechanisms were assessed through hematoxylin-eosin staining, Masson staining, immunostaining, qRT-PCR, and Western blotting. RESULTS: Increased TRPC6 expression was observed in uIR mice and PTECs treated with TGFß1. TRPC6-/- alleviated renal fibrosis by reducing the expression of fibrotic markers (Col-1, α-SMA, and vimentin), as well as decreasing the apoptosis and inflammation of PTECs during fibrotic progression both in vivo and in vitro. Additionally, we found that the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/glycogen synthase kinase 3 beta (GSK3ß) signaling pathway, a pivotal player in renal fibrosis, was down-regulated following TRPC6 deletion. CONCLUSION: These results suggest that the ablation of TRPC6 may mitigate renal fibrosis by inhibiting the apoptosis and inflammation of PTECs through down-regulation of the PI3K/AKT/GSK3ß pathway. Targeting TRPC6 could be a novel therapeutic strategy for preventing chronic kidney disease.

Effect of ADSCs on Th17/Treg and T-bet/GATA-3 in model mice with primary immune thrombocytopenia.

We aimed to observe the effects of adipose-derived mesenchymal stem cells (ADSCs) on T helper 17 (Th17)/regulatory T cells (Treg) and T-box transcription factor (T-bet)/GATA-binding protein 3 (GATA-3) in model mice with primary immune thrombocytopenia (ITP). 32 BALB/C mice were selected. ADSCs were isolated from 2 mice and cultured. The other 30 mice were randomly divided into the normal control group, the ITP model control group, and the ITP experimental group. Platelet count (PLT), Th17/Treg cells, related serum cytokines [interleukin-6 (IL-6), IL-17A, IL-10, and transforming growth factor ß1 (TGF-ß1)], T-bet and GATA-3 mRNA levels in peripheral blood mononuclear cells (PBMCs) in the 3 groups were detected. PLT and Treg in the ITP experimental group were significantly lower than those in the normal control group (P<0.05), but significantly higher than those in the ITP model control group (P<0.05). Th17 and Th17/Treg in the ITP experimental group were significantly higher than those in the normal control group (P<0.05), but significantly lower than those in the ITP model control group (P<0.05). Serum IL-6 and IL-17A levels, and T-bet mRNA levels in the ITP experimental group were significantly higher than those in the normal control group (P<0.05), but significantly lower than those in the ITP model control group (P<0.05). Serum IL-10 and TGF-ß levels, and GATA-3 mRNA levels in the ITP experimental group were significantly lower than those in the normal control group (P<0.05), but significantly higher than those in the ITP model control group (P<0.05). ADSCs can effectively regulate Th17/Treg balance and improve T-bet/GATA-3 mRNA expression levels in ITP model mice.

[Study on mitigating effect and mechanism of Cichorium glandulosum n-butanol extraction site on CCl_4-induced chronic liver injury in rats].

This study aims to investigate the mitigating effect and mechanism of Cichorium glandulosum n-butanol extraction site(CGE) on the disease in carbon tetrachloride(CCl_4)-induced chronic liver injury model in rats. A chronic liver injury model was constructed by subcutaneous injection of CCl_4 olive oil solution, and after four weeks of CGE treatment, serum levels of aspartate aminotransferase(AST), alanine aminotransferase(ALT), alkaline phosphatase(AKP), hydroxyproline(HYP), interleukin-4(IL-4), interleukin-6(IL-6), malondialdehyde(MDA), superoxide dismutase(SOD), and tumor necrosis factor-α(TNF-α) were detected. Liver tissue was processed by hematoxylin-eosin(HE) staining and Masson staining to observe the structure of the rat liver. qPCR and Western blot were used to examine the expression of transforming growth factor-ß1(TGF-ß1)/small mothers against decapentaplegic(Smad), Toll-like receptor 4(TLR4), α-smooth muscle actin(α-SMA), and fibronectin(Fn) in rat liver tissue and hepatic stellate-T6(HSC-T6) and evaluate the inhibitory effect of CGE on HSC activation. The results showed that CGE could significantly reduce the serum levels of AST, ALT, AKP, HYP, and affect the levels of related inflammatory indexes including IL-4, IL-6, and TNF-α, and MDA in CCl_4-induced chronic liver injury in rats and had no effect on SOD activity, which could delay the process of liver injury, alleviate the hepatic collagen deposition and inflammatory infiltration, and had significant efficacy in mitigating chronic liver injury in rats. CGE could inhibit α-SMA and TLR4 protein expression in the liver tissue and reverse the increased TGF-ß1/Smad, Fn, and TLR4-related expression in HSC-T6 in vitro. The above results indicated that CGE exerted hepatoprotective effects in rats by inhibiting HSC activation and alleviated CCl_4-induced chronic liver injury in rats and could ameliorate inflammatory response and slight liver fibrosis in rat liver tissue. Its pharmacodynamic mechanism might be related to TGF-ß1/Smad and TLR4-related expression.

[Mechanism of Fuzheng Huayu Tablets against hepatic fibrosis based on transcriptome and single-cell sequencing].

This study aimed to investigate the role of macrophage polarization in the treatment of liver fibrosis by Fuzheng Huayu Tablets(FZHY) through single-cell, transcriptome sequencing and in vitro and in vivo experiments. Liver fibrosis-related datasets, transcriptomic datasets, and single-cell sequencing datasets were obtained from the Gene Expression Omnibus(GEO) database to screen differential genes. Liver fibrosis-related genes were obtained from GeneCards, DisGeNET, NCBI, PharmgKB, TTD and OMIM databases. Macrophage polarization-related genes were obtained from the GeneCards database. The above three gene sets were intersected to construct a protein-protein interaction(PPI) network. Cytoscape software was used to screen core proteins, and the expression pattern of core proteins was visualized by single-cell sequencing. A mouse model of liver fibrosis was constructed using carbon tetrachloride(CCl_4). Hematoxylin-eosin(HE) staining and Masson staining were used to observe the pathological morphology of liver tissues. The expressions of α-smooth muscle actin(α-SMA) and transforming growth factor-ß1(TGF-ß1) were detected by immunohistochemistry. The levels of alanine aminotransferase(ALT) and aspartate aminotransferase(AST) were detected by colorimetry. The le-vels of inflammatory factors in serum were detected by the enzyme-linked immunosorbent assay(ELISA). Furthermore, the expressions of α-SMA, TGF-ß1, cluster of differentiation 86(CD86) and thrombospondin 1(THBS1) in liver tissues were detected by Western blot(WB). Lipopolysaccharide(LPS) was used to stimulate RAW264.7 cells to construct the M1 macrophage polarization model. The cell counting kit-8(CCK-8) method was used to detect cell viability. WB was used to detect the protein expressions of CD86 and THBS1 in cells, and the messenger ribonucleic acid(mRNA) expression levels of tumor necrosis factor-α(TNF-α) and interleukin(IL)-1ß by real-time fluorescent quantitative reverse transcription polymerase chain reaction(RT-qPCR). The results showed that a total of 26 potential genes related to the polarization of liver fibrosis macrophages were obtained, and 10 core proteins related to the polarization of liver fibrosis macrophages such as THBS1, lumican(LUM) and fibulin-5(FBLN5) were screened. Single-cell data analysis indicated that THBS1, ranking highest, may be expressed by M1 macrophages. Animal experiments demonstrated that FZHY reduced inflammatory cell infiltration and collagen deposition in CCl_4-induced mouse liver, relieved liver injury and inflammation levels, and inhibited the expressions of α-SMA, TGF-ß1, CD86, and THBS1 proteins. Cell experiments revealed that FZHY significantly reduced intracellular expression of CD86 and THBS1 proteins and mRNA levels of TNF-α and IL-1ß. In conclusion, FZHY may ameliorate liver fibrosis by inhibiting THBS1 protein expression, suppressing M1 macrophage polarization, and reducing inflammation.

Novel Sesquiterpenoids with Renoprotective Activities from the Fruits of Alpinae oxyphylla as Potent TGF-ß1/Smads Phosphorylation Inhibitors.

The fruit of Alpinia oxyphylla Miq is an important food spice in southern China and has been used in the treatment of kidney disorders for centuries. In order to discover the natural products with potent renoprotective activities in A. oxyphylla and provide some references for its usage, systematic phytochemical studies were carried out and 24 new diverse sesquiterpenoids, including seven guaiane sesquiterpenoids (1-7), 10 eudesmane sesquiterpenoids (9-13, 18, 19, and 21-23), six cadinane sesquiterpenoids (31-35 and 38), and an eremophilane sesquiterpenoid (40), along with 24 known analogues were isolated and elucidated by analysis of spectroscopic data and quantum-chemical calculations. Biological evaluation showed that 6 sesquiterpenoids could significantly inhibit the expression of extracellular matrix components, α-SMA in TGF-ß1 induced kidney proximal tubular cells (NRK-52e) at low concentrations, and 9 sesquiterpenoids could also downregulate fibronectin and collagen I in a concentration-dependent manner, showing their potential in renal fibrosis. Further action mechanism study displayed that TGF-ß1/Smads pathway might be involved in the antifibrotic effects of active sesquiterpenoids 15 and 43. These studies suggest that A. oxyphylla may have a potential to serve as a functional food in preventing renal fibrosis-associated diseases.

Exposure to BPA and BPS during pregnancy disrupts the bone mineralization in the offspring.

Exposure to plastic-derived estrogen-mimicking endocrine-disrupting bisphenols can have a long-lasting effect on bone health. However, gestational exposure to bisphenol A (BPA) and its analogue, bisphenol S (BPS), on offspring's bone mineralization is unclear. The effects of in-utero bisphenol exposure were examined on the offspring's bone parameters. BPA and BPS (0.0, 0.4 µg/kg bw) were administered to pregnant Wistar rats via oral gavage from gestational day 4-21. Maternal exposure to BPA and BPS increased bone mineral content and density in the offspring aged 30 and 90 days (P < 0.05). Plasma analysis revealed that alkaline phosphatase, and Gla-type osteocalcin were significantly elevated in the BPS-exposed offspring (P < 0.05). The expression of BMP1, BMP4, and their signaling mediators SMAD1 mRNAs were decreased in BPS-exposed osteoblast SaOS-2 cells (P < 0.05). The expression of extracellular matrix proteins such as ALPL, COL1A1, DMP1, and FN1 were downregulated (P < 0.05). Bisphenol co-incubation with noggin decreased TGF-ß1 expression, indicating its involvement in bone mineralization. Altered mineralization could be due to dysregulated expression of bone morphogenetic proteins and signalling mediators in the osteoblast cells. Thus, bisphenol exposure during gestation altered growth and bone mineralization in the offspring, possibly by modulating the expression of Smad-dependent BMP/TGF-ß1 signalling mediators.

Evaluation of differences in expression pattern of three isoforms of the transforming growth factor beta in patients with lumbosacral stenosis.

The study investigates molecular changes in the lumbosacral (L/S) spine's yellow ligamentum flavum during degenerative stenosis, focusing on the role of transforming growth factor beta 1-3 (TGF-ß-1-3). Sixty patients with degenerative stenosis and sixty control participants underwent molecular analysis using real-time quantitative reverse transcription reaction technique (RTqPCR), enzyme-linked immunosorbent assay (ELISA), Western blot, and immunohistochemical analysis (IHC). At the mRNA level, study samples showed reduced expression of TGF-ß-1 and TGF-ß-3, while TGF-ß-2 increased by only 4%. Conversely, at the protein level, the study group exhibited significantly higher concentrations of TGF-ß-1, TGF-ß-2, and TGF-ß-3 compared to controls. On the other hand, at the protein level, a statistically significant higher concentration of TGF-ß-1 was observed (2139.33 pg/mL ± 2593.72 pg/mL vs. 252.45 pg/mL ± 83.89 pg/mL; p < 0.0001), TGF-ß-2 (3104.34 pg/mL ± 1192.74 pg/mL vs. 258.86 pg/mL ± 82.98 pg/mL; p < 0.0001), TGF-ß-3 (512.75 pg/mL ± 107.36 pg/mL vs. 55.06 pg/mL ± 9.83 pg/mL, p < 0.0001) in yellow ligaments obtained from patients of the study group compared to control samples. The study did not establish a significant correlation between TGF-ß-1-3 concentrations and pain severity. The findings suggest that molecular therapy aimed at restoring the normal expression pattern of TGF-ß-1-3 could be a promising strategy for treating degenerative stenosis of the L/S spine. The study underscores the potential therapeutic significance of addressing molecular changes at the TGF-ß isoforms level for better understanding and managing degenerative spinal conditions.

[Study of metal organic framework with siRNA for overcoming matrix barrierin breast cancer].

Objective: This study aimed to develop a new delivery strategy that utilized metal organic framework (MOF) loaded with small-interfering RNA (siRNA) targeting ITGAV to overcome tumor matrix barrier, and thus enhance drug penetration and immune accessibility in breast cancer. Methods: MOF@siITGAV particles were constructed and characterized. The uptake of MOF@siITGAV in breast cancer cell line 4T1 was observed by the cellular uptake assay. The toxicity of MOF@siITGAV was detected by cell counting kit 8 (CCK-8). The blank control group, naked siITGAV group and MOF@siITGAV group were set. Real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) and Western blot were used to detect the expressions of ITGAV. The level of transforming growth factor ß1 (TGF-ß1) in the cell culture medium was detected by enzyme-linked immunosorbent assay (ELISA). The penetration of MOF@siITGAV in 4T1 cells was tested by constructing 3D spheroids. Mouse models of triple negative breast cancer were established. The effect of MOF@siITGAV on the growth of transplanted tumors and main organs was verified. Imminohistochemical (IHC) was used to test the expression of collagen and CD8. Results: MOF@siITGAV particles were constructed with sizes of (198.0±3.3) nm and zeta potential of -(20.2±0.4) mV. MOF@siITGAV could be engulfed by 4T1 cells and triggered to release siRNA. Compared to the blank control group, the expression of ITGAV in the MOF@siITGAV group [(46.5±11.3)%] and the naked siITGAV group [(109.9±19.0)%] was lower. TGF-ß1 in the cell culture medium of the blank control group, naked siITGAV group, and MOF@siITGAV group was (474.5±34.4) pg/ml, (437.2±16.5) pg/ml, and (388.4±14.4) pg/ml, respectively. MOF@siITGAV could better penetrate into 4T1 spheroids and exhibit no obvious toxicity. The cell viability was (99.7±3.5)%, (98.2±5.2)%, (97.3±6.6)%, (92.1±8.1)%, and (92.4±4.1)%, respectively, after MOF@siITGAV treatment with the concentration of 0, 10, 20, 40, 80, and 160 µg/ml, respectively, for 24 h. The tumor growth in the MOF@siITGAV group was suppressed significantly. After 15-day treatment, the tumor volume of the MOF@siITGAV group was (135.3±41.9) mm3, smaller than that of the blank control group [(691.1±193.0) mm3] (P=0.025). The expression of collagen and the number of CD8 positive cells of the MOF@siITGAV group were lower than those of the other two groups. No significant abnormalities were observed in the main organs of mice. Conclusions: Targeting the integrinαv on the surface of cancer cells could destroy extracellular matrix, improve drug delivery, and increase immune infiltration.

The mitigating effect of para-hydroxycinnamic acid in bleomycin-induced pulmonary fibrosis in mice through targeting oxidative, inflammatory and fibrotic pathways.

This study investigated the therapeutic benefits of para-hydroxycinnamic acid in mice with bleomycin-induced lung fibrosis. Forty male BALB/c mice were randomly assigned to four groups: normal, which received 0.9% normal saline; induced, which received a single dose of bleomycin (5 mg/kg) by oropharyngeal challenge; pirfenidone-treated; and para-hydroxycinnamic acid-treated, which challenged with bleomycin and received a daily oral dose of 300 and 50 mg/kg, respectively, from day 7 to day 21. Tissue pro-fibrotic and inflammatory cytokines, oxidative indicators, pulmonary histopathology, immunohistochemistry of fibrotic proteins and the assessment of gene expression by RT-qPCR were evaluated on day 22 after euthanizing animals. Pirfenidone and para-hydroxycinnamic acid managed to alleviate the fibrotic endpoints by statistically improving the weight index, histopathological score and reduced expression of fibrotic-related proteins in immune-stained lung sections, as well as fibrotic markers measured in serum samples. They also managed to alleviate tissue levels of oxidative stress and inflammatory and pro-fibrotic mediators. para-Hydroxycinnamic acid enhanced the expression of crucial genes associated with oxidative stress, inflammation and fibrosis in vivo. para-Hydroxycinnamic acid has demonstrated similar effectiveness to pirfenidone, suggesting it could be a promising treatment for fibrotic lung conditions by inhibiting the TGF-ß1/Smad3 pathway or through its anti-inflammatory and antioxidant properties.

Hsa_circ_0009096/miR-370-3p modulates hepatic stellate cell proliferation and fibrosis during biliary atresia pathogenesis.

Background: Hepatic stellate cell (HSC) activation and hepatic fibrosis mediated biliary atresia (BA) development, but the underlying molecular mechanisms are poorly understood. This study aimed to investigate the roles of circRNA hsa_circ_0009096 in the regulation of HSC proliferation and hepatic fibrosis. Methods: A cellular hepatic fibrosis model was established by treating LX-2 cells with transforming growth factor ß (TGF-ß1). RNaseR and actinomycin D assays were performed to detect hsa_circ_0009096 stability. Expression of hsa_circ_0009096, miR-370-3p, and target genes was detected using reverse transcription-qPCR. Direct binding of hsa_circ_0009096 to miR-370-3p was validated using dual luciferase reporter assay. Cell cycle progression and apoptosis of LX-2 cells were assessed using flow cytometry. The alpha-smooth muscle actin (α-SMA), collagen 1A1 (COL1A1), and TGF beta receptor 2 (TGFBR2) protein levels in LX-2 cells were analyzed using immunocytochemistry and western blotting. Results: Hsa_circ_0009096 exhibited more resistance to RNase R and actinomycinD digestion than UTRN mRNA. Hsa_circ_0009096 expression increased significantly in LX-2 cells treated with TGF-ß1, accompanied by elevated α-SMA and COL1A1 expression. Hsa_circ_0009096 siRNAs effectively promoted miR-370-3p and suppressed TGFBR2 expression in LX-2 cells, mediated by direct association of hsa_circ_0009096 with miR-370-3p. Hsa_circ_0009096 siRNA interfered with the cell cycle progression, promoted apoptosis, and reduced α-SMA and COL1A1 expression in LX-2 cells treated with TGF-ß1. MiR-370-3p inhibitors mitigated the alterations in cell cycle progression, apoptosis, and α-SMA, COL1A1, and TGFBR2 expression in LX-2 cells caused by hsa_circ_0009096 siRNA. In conclusion, hsa_circ_0009096 promoted HSC proliferation and hepatic fibrosis during BA pathogenesis by accelerating TGFBR2 expression by sponging miR-370-3p.

Time-course and muscle-specific gene expression of matrix metalloproteinases and inflammatory cytokines in response to acute treadmill exercise in rats.

BACKGROUND: The extracellular matrix (ECM) of skeletal muscle plays a pivotal role in tissue repair and growth, and its remodeling tightly regulated by matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), and inflammatory cytokines. This study aimed to investigate changes in the mRNA expression of MMPs (Mmp-2 and Mmp-14), TIMPs (Timp-1 and Timp-2), and inflammatory cytokines (Il-1ß, Tnf-α, and Tgfß1) in the soleus (SOL) and extensor digitorum longus (EDL) muscles of rats following acute treadmill exercise. Additionally, muscle morphology was examined using hematoxylin and eosin (H&E) staining. METHODS AND RESULTS: Male rats were subjected to acute treadmill exercise at 25 m/min for 60 min with a %0 slope. The mRNA expression of ECM components and muscle morphology in the SOL and EDL were assessed in both sedentary and exercise groups at various time points (immediately (0) and 1, 3, 6, 12, and 24 h post-exercise). Our results revealed a muscle-specific response, with early upregulation of the mRNA expression of Mmp-2, Mmp-14, Timp-1, Timp-2, Il-1ß, and Tnf-α observed in the SOL compared to the EDL. A decrease in Tgfß1 mRNA expression was evident in the SOL at all post-exercise time points. Conversely, Tgfß1 mRNA expression increased at 0 and 3 h post-exercise in the EDL. Histological analysis also revealed earlier cell infiltration in the SOL than in the EDL following acute exercise. CONCLUSIONS: Our results highlight how acute exercise modulates ECM components and muscle structure differently in the SOL and EDL muscles, leading to distinct muscle-specific responses.

SIRT2-dependent DKK1 deacetylation aggravates polycystic ovary syndrome by targeting the TGF-ß1/Smad3 signaling pathway.

BACKGROUND: Polycystic ovarian syndrome (PCOS) is a prevalent metabolic and endocrine condition in females of reproductive age. This work was to discover the underlying role of Dickkopf 1 (DKK1) and its putative regulating mechanism in P COS. METHODS: Mice recieved dehydroepiandrosterone (DHEA) injection to establish the in vivo P COS model.Hematoxylin and eosin (H&E) staining was performed for histological analysis. RT-qP CR and Western blotting were used to detect gene and protein expression. CCK-8 and flow cytometry assays were applied to detect cell viability and apoptosis. Co-immunoprecipitation (Co-IP) and immunoprecipitation (IP) were applied to assess association between DKK1 and SIRT2. RESULTS: In this work, DKK1 is downregulated in P COS rats. It was revealed that DKK1 knockdown induced apoptosis and suppressed proliferation in KGN cells, whereas DKK1 overexpression had exactly the opposite effects. In addition, DKK1 deactivates the T GF-ß1/SMad3 signaling pathway, thereby controlling KGN cell proliferation and apoptosis. Besides, SIRT2 inhibition reversed the impact of DKK1 overexpression on KGN cell proliferation and apoptosis. Furthermore, SIRT2 downregulated DKK1 expression by deacetylating DKK1 in KGN cells. DISCUSSION: Altogether, we concluded that SIRT2-induced deacetylation of DKK1 triggers T GF-ß1/Smad3 hyperactivation, thereby inhibiting proliferation and promoting apoptosis of KGN cells. The above results indicated that DKK1 might function as a latent target for P COS treatment.