Hydrostatic pressure mediates epithelial-mesenchymal transition of cholangiocytes through RhoA/ROCK and TGF-ß/smad pathways.

Biomechanical cue within the tissue microenvironment is known to play a critical role in regulating cell behaviors and maintaining tissue homeostasis. As hydrostatic pressure often increases in biliary system under pathological states, we investigated the effect of the moderate elevation of the hydrostatic pressure on biliary epithelial cells, especially on the epithelial-mesenchymal transition (EMT). Human intrahepatic biliary epithelial cells were loaded to hydrostatic pressure using a commercial device. We found that loading the cells to 50 mmHg hydrostatic pressure induced obvious morphological changes and significantly upregulated vimentin, ZEB1, and pSmad2/3, fibronectin, and collagen 1α. All changes induced by hydrostatic pressure loading were effectively mitigated by either ROCK inhibitor (Y-27632) or ALK5 inhibitor (SB-431542). Our in vitro experimental data suggests that hydrostatic pressure loading induces EMT of cholangiocytes through RhoA/ROCK and TGF-ß/Smad pathways. Elevated hydrostatic pressure in biliary duct system under pathological states may promote the biliary epithelial cells shifting to profibrotic and mesenchymal characteristics.

Combination of losartan with pirfenidone: a protective anti-fibrotic against pulmonary fibrosis induced by bleomycin in rats.

Pirfenidone (PFD), one acceptable medication for treating idiopathic pulmonary fibrosis (IPF), is not well tolerated by patients at full doses. Hence, employing of some approaches such as combination therapy may be applicable for increasing therapeutic efficacy of PFD. Losartan (LOS), an angiotensin II receptor antagonist, could be a suitable candidate for combination therapy because of its stabilizing effect on the pulmonary function of IPF patients. Therefore, this study aimed to investigate the effects of LOS in combination with PFD on bleomycin (BLM)-induced lung fibrosis in rats. BLM-exposed rats were treated with LOS alone or in combination with PFD. The edema, pathological changes, level of transforming growth factor-ß (TGF-ß1), collagen content, and oxidative stress parameters were assessed in the lung tissues. Following BLM exposure, the inflammatory response, collagen levels, and antioxidant markers in rat lung tissues were significantly improved by PFD, and these effects were improved by combination with LOS. The findings of this in vivo study suggest that the combined administration of PFD and LOS may provide more potent protection against IPF than single therapy through boosting its anti-inflammatory, anti-fibrotic, and anti-oxidant effects. These results hold promise in developing a more effective therapeutic strategy for treating of lung fibrosis.

ELABELA-derived peptide ELA13 attenuates kidney fibrosis by inhibiting the Smad and ERK signaling pathways. / ELABELA衍生肽ELA13通过抑制Smad和ERK信号通路减轻肾纤维化.

Kidney fibrosis is an inevitable result of various chronic kidney diseases (CKDs) and significantly contributes to end-stage renal failure. Currently, there is no specific treatment available for renal fibrosis. ELA13 (amino acid sequence: RRCMPLHSRVPFP) is a conserved region of ELABELA in all vertebrates; however, its biological activity has been very little studied. In the present study, we evaluated the therapeutic effect of ELA13 on transforming growth factor-ß1 (TGF-ß1)-treated NRK-52E cells and unilateral ureteral occlusion (UUO) mice. Our results demonstrated that ELA13 could improve renal function by reducing creatinine and urea nitrogen content in serum, and reduce the expression of fibrosis biomarkers confirmed by Masson staining, immunohistochemistry, real-time polymerase chain reaction (RT-PCR), and western blot. Inflammation biomarkers were increased after UUO and decreased by administration of ELA13. Furthermore, we found that the levels of essential molecules in the mothers against decapentaplegic (Smad) and extracellular signal-regulated kinase (ERK) pathways were reduced by ELA13 treatment in vivo and in vitro. In conclusion, ELA13 protected against kidney fibrosis through inhibiting the Smad and ERK signaling pathways and could thus be a promising candidate for anti-renal fibrosis treatment.

The Effect of a Rotating Magnetic Field on the Regenerative Potential of Platelets.

Platelets are actively involved in tissue injury site regeneration by producing a wide spectrum of platelet-derived growth factors such as PDGF (platelet-derived growth factor), IGF-1 (insulin-like growth factor), TGF-ß1 (transforming growth factor ß), FGF (fibroblast growth factor), etc. A rotating magnetic field (RMF) can regulate biological functions, including reduction or induction regarding inflammatory processes, cell differentiation, and gene expression, to determine the effect of an RMF on the regenerative potential of platelets. The study group consisted of 30 healthy female and male volunteers (n = 15), from which plasma was collected. A portion of the plasma was extracted and treated as an internal control group. Subsequent doses of plasma were exposed to RMF at different frequencies (25 and 50 Hz) for 1 and 3 h. Then, the concentrations of growth factors (IGF-1, PDGF-BB, TGF-ß1, and FGF-1) were determined in the obtained material by the ELISA method. There were statistically significant differences in the PDGF-BB, TGF-ß1, IGF-1, and FGF-1 concentrations between the analyzed groups. The highest concentration of PDGF-BB was observed in the samples placed in RMF for 1 h at 25 Hz. For TGF-ß1, the highest concentrations were obtained in the samples exposed to RMF for 3 h at 25 Hz and 1 h at 50 Hz. The highest concentrations of IGF-1 and FGF-1 were shown in plasma placed in RMF for 3 h at 25 Hz. An RMF may increase the regenerative potential of platelets. It was noted that female platelets may respond more strongly to RMF than male platelets.

Adenosine increases PD-L1 expression in mesenchymal stromal cells derived from cervical cancer through its interaction with A2AR/A2BR and the production of TGF-ß1.

Mesenchymal stromal cells (MSCs) together with malignant cells present in the tumor microenvironment (TME), participate in the suppression of the antitumor immune response through the production of immunosuppressive factors, such as transforming growth factor beta 1 (TGF-ß1). In previous studies, we reported that adenosine (Ado), generated by the adenosinergic activity of cervical cancer (CeCa) cells, induces the production of TGF-ß1 by interacting with A2AR/A2BR. In the present study, we provide evidence that Ado induces the production of TGF-ß1 in MSCs derived from CeCa tumors (CeCa-MSCs) by interacting with both receptors and that TGF-ß1 acts in an autocrine manner to induce the expression of programmed death ligand 1 (PD-L1) in CeCa-MSCs, resulting in an increase in their immunosuppressive capacity on activated CD8+ T lymphocytes. The addition of the antagonists ZM241385 and MRS1754, specific for A2AR and A2BR, respectively, or SB-505124, a selective TGF-ß1 receptor inhibitor, in CeCa-MSC cultures significantly inhibited the expression of PD-L1. Compared with CeCa-MSCs, MSCs derived from normal cervical tissue (NCx-MSCs), used as a control and induced with Ado to express PD-L1, showed a lower response to TGF-ß1 to increase PD-L1 expression. Those results strongly suggest the presence of a feedback mechanism among the adenosinergic pathway, the production of TGF-ß1, and the induction of PD-L1 in CeCa-MSCs to suppress the antitumor response of CD8+ T lymphocytes. The findings of this study suggest that this pathway may have clinical importance as a therapeutic target.

Combination of Fushengong decoction with Western medicine on patients with chronic renal failure: An observational study.

Chronic renal failure (CRF) causes a reduction in glomerular filtration rate and damage to renal parenchyma. Fushengong decoction (FSGD) showed improvement in renal function in CRF rats. This study aims to analyze the differentially expressed proteins in CRF patients treated with Western medicine alone or in combination with FSGD. Sixty patients with CRF recruited from Yongchuan Traditional Chinese Medicine Hospital affiliated to Chongqing Medical University were randomly assigned into control (treated with Western medicine alone) and observation groups (received additional FSGD treatment thrice daily for 8 weeks). The clinical efficacy and changes in serum Bun, serum creatinine, Cystatin C, and transforming growth factor beta 1 (TGF-ß1) before and after treatment were observed. We employed isotope relative labeling absolute quantification labeling and liquid chromatography-mass spectrometry to identify differentially expressed proteins and carried out bioinformatics Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses. Patients in the observation group showed greater clinical improvement and lower levels of serum Bun, serum creatinine, Cyc-c, and TGF-ß1 than the control group. We identified 32 differentially up-regulated and 52 down-regulated proteins in the observation group. These proteins are involved in the blood coagulation system, protein serine/threonine kinase activity, and TGF-ß, which are closely related to the pathogenesis of CRF. Protein-protein-interaction network analysis indicated that candidate proteins fibronectin 1, fibrinogen alpha chain, vitronectin, and Serpin Family C Member 1 were in the key nodes. This study provided an experimental basis suggesting that FSGD combined with Western medicine could significantly improve renal function and renal fibrosis of CRF patients, which may be through the regulation of fibronectin 1, fibrinogen alpha chain, vitronectin, Serpin Family C Member 1, TGF-ß, and the complement coagulation pathway (see Graphical abstract S1, Supplemental Digital Content, http://links.lww.com/MD/L947).

IL-10 and TGF-ß1 may weaken the efficacy of preoperative anti-tuberculosis therapy in older patients with spinal tuberculosis.

Spinal tuberculosis is a common extrapulmonary type that is often secondary to pulmonary or systemic infections. Mycobacterium tuberculosis infection often leads to the balance of immune control and bacterial persistence. In this study, 64 patients were enrolled and the clinicopathological and immunological characteristics of different age groups were analyzed. Anatomically, spinal tuberculosis in each group mostly occurred in the thoracic and lumbar vertebrae. Imaging before preoperative anti-tuberculosis therapy showed that the proportion of abscesses in the older group was significantly lower than that in the younger and middle-aged groups. However, pathological examination of surgical specimens showed that the proportion of abscesses in the older group was significantly higher than that in the other groups, and there was no difference in the granulomatous inflammation, caseous necrosis, inflammatory necrosis, acute inflammation, exudation, granulation tissue formation, and fibrous tissue hyperplasia. B cell number was significantly lower in the middle-aged and older groups compared to the younger group, while the number of T cells, CD4+ T cells, CD8+ T cells, macrophages, lymphocytes, plasma cells, and NK cells did not differ. Meaningfully, we found that the proportion of IL-10 high expression and TGF-ß1 positive in the older group was significantly higher than that in the younger group. TNF-α, CD66b, IFN-γ, and IL-6 expressions were not different among the three groups. In conclusion, there are some differences in imaging, pathological, and immune features of spinal tuberculosis in different age groups. The high expression of IL-10 and TGF-ß1 in older patients may weaken their anti-tuberculosis immunity and treatment effectiveness.

LncRNA GAS5 restrains ISO-induced cardiac fibrosis by modulating mir-217 regulation of SIRT1.

Considering the effect of SIRT1 on improving myocardial fibrosis and GAS5 inhibiting occurrence and development of myocardial fibrosis at the cellular level, the aim of the present study was to investigate whether LncRNA GAS5 could attenuate cardiac fibrosis through regulating mir-217/SIRT1, and whether the NLRP3 inflammasome activation was involved in this process. Isoprenaline (ISO) was given subcutaneously to the male C57BL/6 mice to induce myocardial fibrosis and the AAV9 vectors were randomly injected into the left ventricle of each mouse to overexpress GAS5. Primary myocardial fibroblasts (MCFs) derived from neonatal C57BL/6 mice and TGF-ß1 were used to induce fibrosis. And the GAS5 overexpressed MCFs were treated with mir-217 mimics and mir-217 inhibitor respectively. Then the assays of expression levels of NLRP3, Caspase-1, IL-1ß and SIRT1 were conducted. The findings indicated that the overexpression of GAS5 reduced the expression levels of collagen, NLRP3, Capase-1, IL-1ß and SIRT1 in ISO treated mice and TGF-ß1 treated MCFs. However, this effect was significantly weakened after mir-217 overexpression, but was further enhanced after knockdown of mir-217. mir-217 down-regulates the expression of SIRT1, leading to increased activation of the NLRP3 inflammasome and subsequent pyroptosis. LncRNA GAS5 alleviates cardiac fibrosis induced via regulating mir-217/SIRT1 pathway.

[Metformin suppresses hypoxia-inducible factor-1α expression in cancer-associated fibroblasts to block tumor-stromal cross-talk in breast cancer].

OBJECTIVE: To investigate the mechanism of metformin for regulating tumor-stromal cell cross-talk in breast cancer. METHODS: Tumor associated fibroblasts (CAFs) co-cultured with breast cancer cells were treated with metformin, and the changes in expressions of hypoxia-inducible factor-1α (HIF-1α), p-AMPK, stroma-derived factor-1 (SDF-1) and interleukin-8 (IL-8) in the CAFs were detected using ELISA, RT-qPCR or Western blotting; Transwell assay was used to evaluate the invasiveness of the tumor cells and its changes following treatment with exogenous SDF-1, IL-8 and TGF-ß1. The effects of HIF-1α shRNA or overexpression plasmid, AMPK shRNA, and treatment with OG (a proline hydroxylase inhibitor) or 2-OXO (a proline hydroxylase activator) were examined on p-AMPK, HIF-1α, SDF-1 and IL-8 expressions and invasiveness of the CAFs. RESULTS: Metformin treatment significantly increased the expression levels of p-AMPK, SDF-1 and IL-8 (P<0.05) and decreased HIF-1α expression (P<0.05) without affecting AMPK expression level (P>0.05) in the CAFs. The invasion ability of metformintreated breast cancer cells was significantly decreased (P<0.05). Exogenous SDF-1 and IL-8, HIF-1α overexpression, and OGinduced upregulation of HIF-1α all significantly attenuated the inhibitory effects of metformin on breast cancer cell invasion (P<0.05) and HIF-1α, SDF-1 and IL-8 expressions in CAFs (P<0.05). Transfection with HIF-1α shRNA or treatment with 2-OXO significantly decreased the invasiveness of breast cancer cells (P<0.05). P-AMPK knockdown significantly suppressed the inhibitory effect of metformin on HIF-1α expression in CAFs and on invasion of breast cancer cells (P<0.05). Treatment with TGF-ß1 partially decreased the inhibitory effect of metformin on HIF-1α expression in CAFs and invasiveness of the breast cancer cells (P<0.05). CONCLUSION: Metformin suppresses HIF-1α expression in CAFs to block tumor-stromal cross talk in breast cancer.

[Detection of some markers of the early phase of the inflammatory and reparative process in mechanical skin injuries]. / Vyyavlenie nekotorykh markerov rannei fazy vospalitel'no-reparativnogo protsessa pri mekhanicheskikh povrezhdeniyakh kozhi.

OBJECTIVE: To examine the applicability of IHC staining method: with TGF-ß1 antibodies (serial examination, statistically processed results) and with mast cell tryptase antibodies for injuries vitality determination. MATERIAL AND METHODS: 261 skin autopsy samples with mechanical injuries from 29 persons were divided to 3 groups (87 in each group): vital injuries, postmortal injuries, control non-injured samples. A routine histological examination using standard H&E stain and IHC both with TGF-ß1 and mast cells tryptase antibodies was performed. RESULTS AND CONCLUSION: The positive TGF-ß1 staining (score 2-3) was found in keratinocytes in vitally injured skin and the negative or weak one (score 0-1) was found in control postmortally injured and non-injured samples. Additionally, dermal TGF-ß1 expression was found in some vitally injured skin samples. The difference between vitally injured skin and control samples was statistically significant (p<0.05). No significant difference of dermal mast cells density in groups 1, 2, 3 was found.

Loureirin B ameliorates cholestatic liver fibrosis via AKT/mTOR/ATG7-mediated autophagy of hepatic stellate cells.

AIM OF THE STUDY: Chronic cholestasis leads to liver fibrosis, which lacks effective treatment. In this study, we investigated the role and mechanisms of action of loureirin B (LB) in cholestatic liver fibrosis. MATERIALS AND METHODS: Bile duct ligation (BDL)-induced hepatic fibrosis mice were used as in vivo models. Transforming growth factor-ß1 (TGF-ß1)-pretreated HSC-T6 cells were used to explore the mechanism by which LB attenuates liver fibrosis in vitro. RNA sequencing, quantitative PCR (qPCR), western blotting, immunohistochemistry and immunofluorescence were performed to detect the fibrosis markers and measure autophagy levels. Flow cytometry, cell counting kit-8 (CCK-8) assay, and 5'-ethynyl-2'-deoxyuridine (EdU) assay were conducted to detect cell proliferation and viability. GFP-RFP-LC3 adenovirus, autophagy-related protein 7 (ATG7) siRNA, and bafilomycin A1 (BafA1) were used to verify autophagic flux. RESULTS: Our results showed that LB ameliorates liver injury, inhibits collagen deposition, and decreases the expressions of fibrosis-related markers in BDL-induced mouse livers. In vitro, we found that LB inhibited proliferation and migration, promoted apoptosis, and inhibited the activation of HSC-T6 cells pretreated with TGF-ß1. RNA sequencing analysis of HSC-T6 cells showed that LB treatment predominantly targeted autophagy-related pathways. Further protein analysis indicated that LB downregulated the expression of phosphorylated AKT (p-AKT) and phosphorylated mTOR (p-mTOR), and upregulated LC3-II, p62, and ATG7 both in vivo and in vitro. Intriguingly, ATG7 inactivation reversed the antifibrotic effects of LB on HSC-T6 cells. CONCLUSIONS: LB can improve BDL-induced liver fibrosis by inhibiting the activation and proliferation of HSCs and is expected to be a promising antifibrotic drug.

Silymarin and MSC-exosomes ameliorate thioacetamide-evoked renal fibrosis by inhibiting TGF-ß/SMAD pathway in rats.

BACKGROUND: TGF-ß1 and SMAD3 are particularly pathogenic in the progression of renal fibrosis. AIM: This study aimed to evaluate the kidney protective potentials of silymarin (SM) and exosomes of mesenchymal stem cells against the nephrotoxin thioacetamide (TAA) in rats. METHODS: 32 female rats were randomly assigned into four groups: the control group, the TAA group, the TAA + SM group, and the TAA + Exosomes group. The kidney homogenates from all groups were examined for expression levels of TGF-ß receptors I and II using real-time PCR, expression levels of collagen type I and CTGF proteins using ELISA, and the expression levels of nuclear SMAD2/3/4, cytoplasmic SMAD2/3, and cytoplasmic SMAD4 proteins using the western blot technique. RESULTS: Compared to the control group, the injection of TAA resulted in a significant increase in serum levels of urea and creatinine, gene expression levels of TßRI and TßRII, protein expression levels of both collagen I and CTGF proteins, cytoplasmic SMAD2/3 complex, and nuclear SMAD2/3/4 (p-value < 0.0001), with significantly decreased levels of the co-SMAD partner, SMAD4 (p-value < 0.0001). Those effects were reversed considerably in both treatment groups, with the superiority of the exosomal treatment regarding the SMAD proteins and the expression levels of the TßRI gene, collagen I, and CTGF proteins returning to near-control values (p-value > 0.05). CONCLUSION: Using in vitro and in vivo experimental approaches, the research discovered a reno-protective role of silymarin and exosomes of BM-MSCs after thioacetamide-induced renal fibrosis in rats, with the advantage of exosomes.

F127-SE-tLAP thermosensitive hydrogel alleviates bleomycin-induced skin fibrosis via TGF-ß/Smad pathway.

BACKGROUND: Skin fibrosis affects the normal function of the skin. TGF-ß1 is a key cytokine that affects organ fibrosis. The latency-associated peptide (LAP) is essential for TGF-ß1 activation. We previously constructed and prepared truncated LAP (tLAP), and confirmed that tLAP inhibited liver fibrosis by affecting TGF-ß1. SPACE peptide has both transdermal and transmembrane functions. SPACE promotes the delivery of macromolecules through the stratum corneum into the dermis. This study aimed to alleviate skin fibrosis through the delivery of tLAP by SPACE. METHODS: The SPACE-tLAP (SE-tLAP) recombinant plasmid was constructed. SE-tLAP was purified by nickel affinity chromatography. The effects of SE-tLAP on the proliferation, migration, and expression of fibrosis-related and inflammatory factors were evaluated in TGF-ß1-induced NIH-3T3 cells. F127-SE-tLAP hydrogel was constructed by using F127 as a carrier to load SE-tLAP polypeptide. The degradation, drug release, and biocompatibility of F127-SE-tLAP were evaluated. Bleomycin was used to induce skin fibrosis in mice. HE, Masson, and immunohistochemistry were used to observe the skin histological characteristics. RESULTS: SE-tLAP inhibited the proliferation, migration, and expression of fibrosis-related and inflammatory factors in NIH-3T3 cells. F127-SE-tLAP significantly reduced ECM production, collagen deposition, and fibrotic pathological changes, thereby alleviating skin fibrosis. CONCLUSION: F127-SE-tLAP could increase the transdermal delivery of LAP, reduce the production and deposition of ECM, inhibit the formation of dermal collagen fibers, and alleviate the progression of skin fibrosis. It may provide a new idea for the therapy of skin fibrosis.

The effect of saraglitazar on TGF-ß-induced smad3 phosphorylation and expression of genes related to liver fibrosis in LX2 cell line.

BACKGROUND AND PURPOSE: Liver fibrosis is a reversible liver injury that occurs as a result of many chronic inflammatory diseases and can lead to cirrhosis, which is irreversible and fatal. So, we studied the anti-fibrotic effects of saroglitazar on LX-2 cell lines, as a dual PPARα/γ agonist. METHODS: Cells, after 80% confluence, were treated with TGF-ß (2 ng/mL) for 24 h. Then cells were treated with saroglitazar at different doses (2.5, 5, 10 µM) for 24 h. After same incubation, the cells of control group, TGF-ß group, and TGF-ß + saroglitazar group were harvested for RNA and protein extraction to determine the effects of saroglitazar. RT-PCR and western blot methods were used to express genes related to fibrosis. RESULTS: Our results show that the relative expression of α-SMA, collagen1α, N-cadherin, NOX (1, 2, and 4), and phosphorylated Smad3 protein was significantly higher in TGF-ß-treated cells compared with the normal group, and E-cadherin expression was decreased in TGF-ß-treated cells. After TGF-ß-treated cells were exposed to saroglitazar, the expression of these genes was significantly reversed (P < 0.05). CONCLUSIONS: Our results clearly show the short-term inhibitory role of saroglitazar in the expression of fibrotic factors using the TGF-ß/Smad signaling pathway. These results suggest that saroglitazar can be considered as a suitable therapeutic strategy for fibrotic patients. Although more studies are needed.

[Effects of M2-type macrophages and GKT137831 on oxidative stress in hepatic stellate cells].

Objective: To investigate the effects of reduced nicotinamide adenine dinucleotide phosphooxidase 4 (NOX4) inhibitors GKT137831 and M2-type macrophages on oxidative stress markers NOX4, nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1) in the rat hepatic stellate cell line (HSC-T6). Methods: Rat bone marrow macrophages were extracted and induced using interleukin (IL)-4 to differentiate them into M2 phenotype macrophages. HSC-T6 activation was performed with 5 µg/L transforming growth factor ß1 (TGF-ß1). The proliferation condition of HSC-T6 cells stimulated by the NOX4 inhibitor GKT137831 at a concentration gradient of 5 to 80 µmol/L after 48 hours was detected using the Cell Counting Kit-8 (CCK-8) assay. The optimal drug concentration was chosen and divided into an HSC co-culture group (the control group) and five experimental groups: the TGF-ß1 stimulation group, the TGF-ß1 +GKT137831 stimulation group, the M2-type macrophage + HSC co-culture group, the M2-type macrophage +TGF-ß1 stimulation group, and the M2-type + TGF-ß1 + GKT137831 stimulation group. Reactive oxygen species (ROS) production level was detected in each cell using the DCFH-DA probe method. NOX4, α-smooth muscle actin (α-SMA), Nrf2, and HO-1 levels in each group of HSC cells were detected using the qRT-PCR method and the Western blot method. The t-test was used to compare the two groups. The one-way ANOVA method was used to compare multiple groups. Results: Intracellular ROS increased significantly following TGF-ß1 stimulation. ROS relative levels in each cell group were 1.03±0.11, 3.88±0.07, 2.90±0.08, 0.99±0.06, 3.30±0.05, 2.21±0.11, F = 686.1, P = 0.001, respectively. The mRNA and protein expressions of NOX4, α-SMA, Nrf2, and HO-1 were significantly increased (P < 0.05). After the addition of GKT137831, ROS, and NOX4, α-SMA mRNA and protein expression were comparatively decreased in the TGF-ß1 stimulation group (P < 0.05), while mRNA and protein expressions of Nrf2 and HO-1 were increased (P < 0.05). The expression of ROS and NOX4, as well as α-SMA mRNA and protein, produced by HSC were significantly decreased in the co-culture group compared to the single culture group after TGF-ß1 stimulation (P < 0.05). After the addition of GKT137831, ROS, NOX4, α-SMA mRNA, and protein expression were further reduced in the co-culture group compared with the single culture group (P < 0.05), while the mRNA and protein expression of Nrf2 and HO-1 were further increased (P < 0.05). Conclusion: NOX4 inhibitor GKT137831 can reduce RO, NOX4, and α-SMA levels while increasing Nrf2 and HO-1 levels in hepatic stellate cells. After M2-type macrophage co-culture, GKT137831 assists in lowering ROS, NOX4, and α-SMA levels while accelerating Nrf2 and HO-1 levels in hepatic stellate cells, which regulates the balance between oxidative stress and anti-oxidative stress systems, thereby antagonizing the fibrosis process.

The Effect of Salvianolic Acid A on Tumor-Associated Macrophage Polarization and Its Mechanisms in the Tumor Microenvironment of Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, with a high degree of malignancy and poor prognosis. Tumor-associated macrophages (TAMs) have been identified as significant contributors to the growth and metastasis of TNBC through the secretion of various growth factors and chemokines. Salvianolic acid A (SAA) has been shown to have anti-cancer activities. However, the potential activity of SAA on re-polarized TAMs remains unclear. As there is a correlation between the TAMs and TNBC, this study investigates the effect of SAA on TAMs in the TNBC microenvironment. For that purpose, M2 TAM polarization was induced by two kinds of TNBC-conditioned medium (TNBC-TCM) in the absence or presence of SAA. The gene and protein expression of TAM markers were analyzed by qPCR, FCM, IF, ELISA, and Western blot. The protein expression levels of ERK and p-ERK in M2-like TAMs were analyzed by Western blot. The migration and invasion properties of M2-like TAMs were analyzed by Transwell assays. Here, we demonstrated that SAA increased the expression levels of CD86, IL-1ß, and iNOS in M2-like TAMs and, conversely, decreased the expression levels of Arg-1 and CD206. Moreover, SAA inhibited the migration and invasion properties of M2-like TAMs effectively and decreased the protein expression of TGF-ß1 and p-ERK in a concentration-dependent manner, as well as TGF-ß1 gene expression and secretion. Our current findings for the first time demonstrated that SAA inhibits macrophage polarization to M2-like TAMs by inhibiting the ERK pathway and promotes M2-like TAM re-polarization to the M1 TAMs, which may exert its anti-tumor effect by regulating M1/M2 TAM polarization. These findings highlight SAA as a potential regulator of M2 TAMs and the possibility of utilizing SAA to reprogram M2 TAMs offers promising insights for the clinical management of TNBC.

Longitudinal markers of cerebral amyloid angiopathy and related inflammation in rTg-DI rats.

Cerebral amyloid angiopathy (CAA) is a prevalent vascular dementia and common comorbidity of Alzheimer's disease (AD). While it is known that vascular fibrillar amyloid ß (Aß) deposits leads to vascular deterioration and can drive parenchymal CAA related inflammation (CAA-ri), underlying mechanisms of CAA pathology remain poorly understood. Here, we conducted brain regional proteomic analysis of early and late disease stages in the rTg-DI CAA rat model to gain molecular insight to mechanisms of CAA/CAA-ri progression and identify potential brain protein markers of CAA/CAA-ri. Longitudinal brain regional proteomic analysis revealed increased differentially expressed proteins (DEP) including ANXA3, HTRA1, APOE, CST3, and CLU, shared between the cortex, hippocampus, and thalamus, at both stages of disease in rTg-DI rats. Subsequent pathway analysis indicated pathway enrichment and predicted activation of TGF-ß1, which was confirmed by immunolabeling and ELISA. Further, we identified numerous CAA related DEPs associate with astrocytes (HSPB1 and MLC1) and microglia (ANXA3, SPARC, TGF-ß1) not previously associated with astrocytes or microglia in other AD models, possibly indicating that they are specific to CAA-ri. Thus, the data presented here identify several potential brain protein biomarkers of CAA/CAA-ri while providing novel molecular and mechanistic insight to mechanisms of CAA and CAA-ri pathological progression and glial cell mediated responses.

MiR-132-3p suppresses peritoneal fibrosis induced by peritoneal dialysis via targeting TGF-ß1/Smad2/3 signaling pathway.

BACKGROUND: Peritoneal fibrosis (PF) is the main complication of peritoneal dialysis (PD) and the most common cause of cessation from PD. There is still no effective therapeutic approach to reserve PF. We aimed to investigate the role of miR-132-3p and underlying potential mechanisms in PF. METHODS: A total of 18 Sprague-Dawley (SD) rats were divided randomly into three groups (n = 6): (i)Control group (ii)PF group (iii)PF+Losartan group; Rats in the PF group and PF+Losartan group received daily intraperitoneal injections of 3 mg/kg chlorhexidine for 14 days, and rats in the PF+Losartan group simultaneously received daily intraperitoneal injections of 2 mg/kg losartan for 14 days. The control group was injected with saline in the same volume. Met-5A cells were treated for 24h with TGF-ß1 dissolved in recombinant buffered saline at a concentration of 10 ng/ml, meanwhile, PBS solution as a negative control. The human peritoneal solution was collected for the detection of miR-132-3p. RESULTS: In vivo, SD rats were infused with chlorhexidine to establish PF model, and we found that miR-132-3p significantly decreased and the expressions of transforming growth factor-ß1 (TGF-ß1), and Smad2/3 were up-regulated in PF. In vitro, miR-132-3p mimics suppressed TGF-ß1/Smad2/3 activity, whereas miR-132-3p inhibition activated the pathway. In human peritoneal solution, we found that the expression of miR-132-3p decreased in a time-dependent model and its effect became more pronounced with longer PD duration. CONCLUSION: MiR-132-3p ameliorated PF by suppressing TGF-ß1/Smad2/3 activity, suggesting that miR-132-3p represented a potential therapeutic approach for PF.

Uveal melanoma cell lines Mel270 and 92.1 exhibit a mesenchymal phenotype and sensitivity to the cytostatic effects of transforming growth factor beta in vitro.

Purpose: Uveal melanoma (UM) is a deadly cancer with limited therapeutic options. At advanced stages, UM cells metastasize almost exclusively into the liver, where targeting metastatic UM cells remain a clinical challenge. Transforming growth factor beta (TGF-ß) exhibits a functional duality in cancer, with one arm limiting tumor growth at an early stage and the second arm promoting metastasis at an advanced stage, notably by inducing an epithelial-to-mesenchymal transition. Thus, we hypothesized that targeting the TGF-ß pathway could be relevant in the treatment of metastatic UM. Methods: In this study, we first characterized the pseudoepithelial/mesenchymal phenotype of UM cell lines Mel270 and 92.1. We then treated the cell lines with TGF-ß to evaluate their responsiveness to the cytokine and to characterize the functional impact of TGF-ß on their cell viability. Results: The results demonstrated, first, that the UM cell lines exhibited a mesenchymal phenotype and responded to TGF-ß treatment in vitro and, second, that TGF-ß promoted a cytostatic effect on the UM cell lines. Conclusions: Our findings indicate that UM cells are sensitive to the two arms of TGF-ß signaling, which suggests that targeting the TGF-ß pathway could be challenging in UM and would require a precise selection of patients in which only the prometastatic arm of TGF-ß is activated.

Adipose-derived stem cells promote glycolysis and peritoneal metastasis via TGF-ß1/SMAD3/ANGPTL4 axis in colorectal cancer.

Peritoneal metastasis, the third most common metastasis in colorectal cancer (CRC), has a poor prognosis for the rapid progression and limited therapeutic strategy. However, the molecular characteristics and pathogenesis of CRC peritoneal metastasis are poorly understood. Here, we aimed to elucidate the action and mechanism of adipose-derived stem cells (ADSCs), a prominent component of the peritoneal microenvironment, in CRC peritoneal metastasis formation. Database analysis indicated that ADSCs infiltration was increased in CRC peritoneal metastases, and high expression levels of ADSCs marker genes predicted a poor prognosis. Then we investigated the effect of ADSCs on CRC cells in vitro and in vivo. The results revealed that CRC cells co-cultured with ADSCs exhibited stronger metastatic property and anoikis resistance, and ADSCs boosted the intraperitoneal seeding of CRC cells. Furthermore, RNA sequencing was carried out to identify the key target gene, angiopoietin like 4 (ANGPTL4), which was upregulated in CRC specimens, especially in peritoneal metastases. Mechanistically, TGF-ß1 secreted by ADSCs activated SMAD3 in CRC cells, and chromatin immunoprecipitation assay showed that SMAD3 facilitated ANGPTL4 transcription by directly binding to ANGPTL4 promoter. The ANGPTL4 upregulation was essential for ADSCs to promote glycolysis and anoikis resistance in CRC. Importantly, simultaneously targeting TGF-ß signaling and ANGPTL4 efficiently reduced intraperitoneal seeding in vivo. In conclusion, this study indicates that tumor-infiltrating ADSCs promote glycolysis and anoikis resistance in CRC cells and ultimately facilitate peritoneal metastasis via the TGF-ß1/SMAD3/ANGPTL4 axis. The dual-targeting of TGF-ß signaling and ANGPTL4 may be a feasible therapeutic strategy for CRC peritoneal metastasis.