Expression Of p-Smad2 Research Articles

Abstract 3819: Naringin nanoparticles inhibit metastasis of lung cancer through TGF-β1-smad2/3 signaling pathway

Abstract Lung cancer causes high mortality due to its high metastasis. Naringin is a naturally occurring flavonoid that is widely found in the various citrus fruits. Naringin is known to have several therapeutic effects including anticancer, anti-pulmonary fibrosis, antimicrobial, anti-inflammatory. However, low bioavailability and solubility of naringin limit its application. This project designed a thermosensitive naringin nanoparticle to investigate whether it might inhibit metastasis of lung cancer by inhibiting TGF-β1-smad2/3 signaling pathway. Cells were treated with various concentrations of naringin nanoparticles and unwrapped naringin for 48 hours. Naringin nanoparticles of 100µmol/L showed obvious inhibitory effect on the larger wound surface of cultured human cancer cells. Westerm blot was used to detect the expression of P-Smad2, Smad2,and α-SMA in A549 cells. The results showed that naringin significantly reduced the expression of EMT-related protein α-SMA in A549 cells in a concentration-dependent manner. Naringin nanoparticles can reverse the EMT process. naringin nanoparticles can inhibit the expression of P-Smad2 protein in A549 cells, which may play a role in inhibiting lung cancer through TGF-β1/Smad signaling pathway.The animal experiment was performed. The mice were given various concentrations of naringin nanoparticles and unwrapped naringin by IV. the protein expression levels of α-SMA, TGF-β1 and P-Smad3 in mice lung tissue were detected by immunohistochemistry. The results showed that naringin nanoparticles 50um/ml,0.2mL/day/time had significant effect on the inhibition of lung cancer tissue migration, and the different doses of naringin nanoparticles showed a certain dose-dependent trend. The expression of TGF-β1 and P-Smad3 in mice treated with naringin nanoparticles was significantly decreased, suggesting that naringin nanoparticles could inhibit the migration of lung cancer cells by inhibiting TGF-β1/Smad pathway. Citation Format: Wen Chen. Naringin nanoparticles inhibit metastasis of lung cancer through TGF-β1-smad2/3 signaling pathway. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3819.

Protein phosphatase PPM1A inhibition attenuates osteoarthritis via regulating TGF-β/Smad2 signaling in chondrocytes.

TGF-β signaling is crucial for modulating osteoarthritis (OA), and protein phosphatase magnesium-dependent 1A (PPM1A) has been reported as a phosphatase of SMAD2 and regulates TGF-β signaling, while the role of PPM1A in cartilage homeostasis and OA development remains largely unexplored. In this study, we found increased PPM1A expression in OA chondrocytes and confirmed the interaction between PPM1A and phospho-SMAD2 (p-SMAD2). Importantly, our data show that PPM1A KO substantially protected mice treated with destabilization of medial meniscus (DMM) surgery against cartilage degeneration and subchondral sclerosis. Additionally, PPM1A ablation reduced the cartilage catabolism and cell apoptosis after the DMM operation. Moreover, p-SMAD2 expression in chondrocytes from KO mice was higher than that in WT controls with DMM induction. However, intraarticular injection with SD-208, repressing TGF-β/SMAD2 signaling, dramatically abolished protective phenotypes in PPM1A-KO mice. Finally, a specific pharmacologic PPM1A inhibitor, Sanguinarine chloride (SC) or BC-21, was able to ameliorate OA severity in C57BL/6J mice. In summary, our study identified PPM1A as a pivotal regulator of cartilage homeostasis and demonstrated that PPM1A inhibition attenuates OA progression via regulating TGF-β/SMAD2 signaling in chondrocytes and provided PPM1A as a potential target for OA treatment.

Sildenafil Inhibits the Growth and Epithelial-to-mesenchymal Transition of Cervical Cancer via the TGF-β1/Smad2/3 Pathway.

The study aims to explore new potential treatments for cervical cancer. Cervical cancer is the second most common cancer in women, causing >250,000 deaths worldwide. Patients with cervical cancer are mainly treated with platinum compounds, which often cause severe toxic reactions. Furthermore, the long-term use of platinum compounds can reduce the sensitivity of cancer cells to chemotherapy and increase the drug resistance of cervical cancer. Therefore, exploring new treatment options is meaningful for cervical cancer. The present study was to investigate the effect of sildenafil on the growth and epithelial-tomesenchymal transition (EMT) of cervical cancer. HeLa and SiHa cells were treated with sildenafil for different durations. Cell viability, clonogenicity, wound healing, and Transwell assays were performed. The levels of transforming growth factor-β1 (TGF-β1), transforming growth factor-β type I receptor (TβRI), phosphorylated (p-) Smad2 and p-Smad3 in cervical cancer samples were measured. TGF-β1, Smad2 or Smad3 were overexpressed in HeLa cells, and we measured the expression of EMT marker proteins and the changes in cell viability, colony formation, etc. Finally, HeLa cells were used to establish a nude mouse xenograft model with sildenafil treatment. The survival rate of mice and the tumor size were recorded. High concentrations of sildenafil (1.0-2.0 μM) reduced cell viability, the number of HeLa and SiHa colonies, and the invasion/migration ability of HeLa and SiHa cells in a dose- and time-dependent manner. The expression of TGF-β1, TβRI, p-Smad2 and p-Smad3 was significantly enhanced in cervical cancer samples and cervical cancer cell lines. Sildenafil inhibited the expression of TGF-β1-induced EMT marker proteins (Snail, vimentin, Twist, E-cadherin and N-cadherin) and p-Smad2/3 in HeLa cells. Overexpression of TGF-β1, Smad2, and Smad3 reversed the effect of sildenafil on EMT, viability, colony formation, migration, and invasion ability of HeLa cells. In the in vivo study, sildenafil significantly increased mouse survival rates and suppressed xenograft growth. Sildenafil inhibits the proliferation, invasion ability, and EMT of human cervical cancer cells by regulating the TGF-β1/Smad2/3 pathway.

Gastrodin attenuates renal injury and collagen deposition via suppression of the TGF-β1/Smad2/3 signaling pathway based on network pharmacology analysis.

Background: Gastrodin has been widely used clinically in China as an antihypertensive drug. However, its effect on hypertensive renal injury is yet to be elucidated. The current study aimed to investigate the effects of gastrodin on hypertensive renal injury and its underlying mechanisms by network pharmacology analysis and validation in vivo and in vitro. Methods: A total of 10 spontaneously hypertensive rats (SHRs) were randomly categorized into the following two groups: SHR and SHR + Gastrodin groups. Wistar Kyoto (WKY) rats were used as the control group (n = 5). The SHR + Gastrodin group was intragastrically administered gastrodin (3.5mg/kg/day), and the rats in both WKY and SHR groups were intragastrically administered an equal amount of double-distilled water for 10 weeks. Hematoxylin-eosin, Masson's trichrome, and Sirius red staining were used to detect the pathological changes and collagen content in the renal tissues. Network pharmacology analysis was performed to explore its potential targets and related pathways. In vitro, the CCK-8 assay was used to determine the cell viability. Immunohistochemistry and western-blotting analyses were employed to assess the protein expression associated with renal fibrosis and transforming growth factor-β1 (TGF-β1) pathway-related proteins in the renal tissues or in TGF-β1-stimulated rat kidney fibroblast cell lines (NRK-49F). Results: Gastrodin treatment attenuates renal injury and pathological alterations in SHRs, including glomerular sclerosis and atrophy, epithelial cell atrophy, and tubular dilation. Gastrodin also reduced the accumulation of collagen in the renal tissues of SHRs, which were confirmed by downregulation of α-SMA, collagen I, collagen III protein expression. Network pharmacology analysis identified TGFB1 and SMAD2 as two of lead candidate targets of gastrodin on against hypertensive renal injury. Consistently, gastrodin treatment downregulated the increase of the protein expression of TGF-β1, and ratios of both p-Smad2/Smad2 and p-Samd3/Smad3 in renal tissues of SHRs. In vitro, gastrodin (25-100μM) treatment significantly reversed the upregulation of α-SMA, fibronectin, collagen I, as well as p-Smad2 and p-Smad3 protein expressions without affecting the cell viability of TGF-β1 stimulated NRK-49F cells. Conclusion: Gastrodin treatment significantly attenuates hypertensive renal injury and renal fibrosis and suppresses TGF-β1/Smad2/3 signaling in vivo and in vitro.

Leucine rich repeat containing 32 accelerates tenogenic differentiation of tendon-derived stem cells and promotes Achilles tendon repair in rats.

Although many surgical or non-operative therapies have been developed to treat Achilles tendon injuries, the prognosis of which is often unsatisfactory. Recently, biologic approaches using multipotent stem cells like tendon-derived stem cells (TDSCs) pose a possible treatment option. To evaluate whether the Leucine rich repeat containing 32 (Lrrc32) affects the tenogenic differentiation of TDSCs and thus promotes Achilles tendon healing. TDSCs were infected with the recombinant Lrrc32-overexpressing lentivirus (LV-Lrrc32) and then locally injected into the injured site of rat. Four weeks after surgery, the Achilles tendon tissue (~0.5 cm) around the injured area was harvested for analysis. Pathological results showed that Lrrc32-overexpressing TDSCs significantly improved the morphological changes of the injured tendons. Specifically, the increased collagen-I expression and hydroxyproline content in extracellular matrix, and more orderly arrangement of the regenerated collagen fibers were observed in the Lrrc32 overexpression group. Moreover, 4 weeks after injection of Lrrc32-overexpressing TDSCs, the expression of tenocyte-related genes such as tenomodulin (Tnmd), scleraxis (Scx) and decorin (Dcn) were upregulated in the area of the healing tendon. These findings indicated that Lrrc32 promoted the tenogenic differentiation of TDSCs in vivo. Additionally, Lrrc32 overexpression also increased the expression of TGF-β1 and p-SMAD2/3, suggesting that the beneficial effects of Lrrc32 on tendon repair might be associated with the expression of TGF-β1 and p-SMAD2/3. Our findings collectively revealed that Lrrc32-overexpressed TDSCs promoted tendon healing more effectively than TDSCs alone.

Testosterone attenuates senile cavernous fibrosis by regulating TGFβR1 and galectin-1 signaling pathways through miR-22-3p.

Erectile dysfunction (ED) is a major health problem affecting a large proportion of the general population. Testosterone also plays a key role in sexual dysfunction. In this study, we found that testosterone can inhibit cavernous fibrosis by affecting the expression of miR-22-3p, providing a new basis for research and treatment of ED. Old and young rats were used to study the effects of testosterone on cavernous fibrosis. Hematoxylin and eosin (HE) and Masson's staining were used to observe the cavernous tissue. A luciferase assay was used to analyze the relationship between the miR-22-3p, TGFβR1, and Galectin-1 signaling pathways. CCK-8 and flow cytometry were used to detect the proliferation and apoptosis rates of cavernosum smooth muscle cells (CSMCs) following testosterone intervention. Immunohistochemical analysis was performed to examine the positive rate of caspase 3 and Ki67. IF was used to analyze the expression of collagen IV, MMP2, and α-SMA. The levels of GnRH, tT, LH, and F-TESTO in old rats increased after testosterone intervention. miR-22-3p inhibits the expression of TGFβR1 and Galectin-1. The protein expression of TGFβR1, Galectin-1, SMAD2, and p-SMAD2 was reduced by testosterone. The expression levels of α-SMA, collagen I, collagen IV, FN, and MMP2 in the cavernous tissues of old rats treated with testosterone were significantly reduced. The levels of caspase 3 and collagen IV decreased, and the levels of MMP2, Ki67, and α-SMA increased. Testosterone and miR-22-3p inhibit CSMC apoptosis and promote cell proliferation. Testosterone promoted the expression of miR-22-3p to interfere with the expression of the cavernous TGFβR1 and Galectin-1 signaling pathways. Testosterone can reduce cavernous fibrosis during the treatment of functional ED.

The extract from Hyssopus cuspidatus Boriss. Prevents bronchial airway remodeling by inhibiting mouse bronchial wall thickening and hASMC proliferation and migration

Ethnopharmacological relevanceBronchial asthma, a non-communicable chronic respiratory disease, affects people of all ages. An important pathological feature of bronchial asthma is airway remodeling. Hyssopus cuspidatus Boriss. has been used to treat bronchial asthma for over 100 years in Uygur medicine. The ethanol extract of Hyssopus cuspidatus Boriss.(JAX2) can improve airway inflammation in asthma. However, the anti-asthmatic airway-remodeling effect of JAX2 is unclear. Aim of the studyThe current study investigated the anti-airway remodeling effect of JAX2 and elucidated its mechanism of action. Materials and methodsThe present study established an ovalbumin-induced mouse model of asthma and platelet-derived growth factor-BB-induced human airway smooth muscle cells (hASMCs) proliferation model, with dexamethasone (DEX) and feining tablets (FNP) designated as positive control drugs. Pathological changes in lung tissues were observed using hematoxylin and eosin staining. Interleukin (IL)-5, IL-10, IL-13, and IL-33 levels in the bronchoalveolar lavage fluid (BALF) and serum of mice were determined using enzyme-linked immunosorbent assay (ELISA). Changes in the expression and distribution of TGF-β1, p-ERK1/2, Smad2/3, and p-Smad3 in lung tissues were determined using immunohistochemistry. Western blotting (WB) was used to determine the protein levels of p-ERK1/2 in lung tissues and cells. MTS assay was used to determine the effects of JAX2 on cell proliferation. IL-5, IL-10, IL-13, MMP-2, and MMP-9 levels in the cell supernatant were determined using ELISA. HASMCs migration was observed using the scratch and transwell methods. The effect of JAX2 on the hASMCs cycle was determined using flow cytometry. ResultsJAX2 significantly improved the pathological status of lung tissues in asthmatic mice. It could also significantly reduce IL-5, IL-13, and IL-33 levels in the BALF and serum of asthmatic mice in a dose-dependent manner and significantly increase IL-10 levels. TGF-β1, p-ERK1/2, Smad2/3, and p-Smad3 expression in lung tissues were decreased in a dose-dependent manner. The protein level of p-ERK1/2 in lung tissues was also reduced. JAX2 could significantly inhibit the proliferation and migration of PDGF-BB-induced hASMCs. IL-5, IL-13, MMP-9, and MMP-2 levels decreased significantly, and IL-10 levels increased significantly in a dose-dependent manner in the cell supernatant. JAX2 could block hASMCs in the G0/G1 phase, thereby inhibiting cell proliferation. p-ERK1/2 protein levels were found to decrease in a dose-dependent manner. ConclusionsJAX2 significantly inhibits airway remodeling in asthma. Its mechanism of action may be inhibiting the proliferation and migration of hASMCs, releasing inflammatory factors and metalloproteinases, activating the ERK1/2 signal pathway, and promoting the secretion of anti-inflammatory factors.

Endosulfan induced kidney cell injury by modulating ACE2 through up-regulating miR-429 in HK-2 cells

Endosulfan, a typical organochlorine pesticide, is widely used in agricultural countries and was detected in blood samples from the general population. Studies have shown a positive correlation between chronic kidney disease of unknown aetiology (CKDu) and endosulfan. CKDu has become endemic in agricultural countries, with clinical manifestations of tubulointerstitial fibrosis.The goal of this study was to investigate the effects of endosulfan in kidney cell injury in human renal tubular epithelial cells (HK-2), focusing on apoptosis, inflammatory response, and epithelial-mesenchymal transition (EMT). We found that endosulfan induced apoptosis in HK-2 cells by up-regulating the expression of BAX, APAF-1, Caspase-3 and mitochondrial Cytochrome c was released into the cytosol. Endosulfan caused an inflammatory response, showing the increase in the secretion and mRNA expression levels of IL-6/IL-8. Endosulfan triggered EMT, characterized by downregulation of E-cadherin and upregulation of Vimentin. Western blot results showed that p-Smad3 and Smad3 protein expression were elevated while the expression of Smad7 were decreased in endosulfan-exposed groups. Dual luciferase reporter assay confirmed the potential binding capacity of miR-429 to 3'-UTR of ACE2. Endosulfan causes upregulation of miR-429 and downregulation of ACE2 in HK-2 cells. Overexpression of miR-429 or silencing of ACE2 in HK-2 cells caused apoptosis, inflammation and EMT through TGF signaling pathway. These findings suggest that endosulfan can lead to kidney cell injury by modulating ACE2 through up-regulating miR-429, providing new evidence for the pathogenesis of CKDu.

MicroRNA-29a inhibits collagen expression and induces apoptosis in human fetal scleral fibroblasts by targeting the Hsp47/Smad3 signaling pathway.

Members of the microRNA-29 (miR-29) gene family have been implicated as suppressors of collagen in several human diseases. The present study aimed to explore the function of miR-29a in human fetal scleral fibroblasts (HFSFs) and to investigate potential mechanisms by which the molecule regulates cellular functioning. First, HFSFs were transfected with miR-29a mimic, miR-29a inhibitor, or their corresponding controls. Then, cell proliferation and apoptosis were assessed using a CCK-8 assay and flow cytometry, respectively. Further, using real-time PCR, western blotting, and immunofluorescence staining, levels of miR-29a, heat shock protein 47 (Hsp47), COL1A1, Smad3, P-Smad3, Bax, and Bcl-2 were investigated. Next, empty vectors and SERPINH1-overexpressing vectors were used to transfect HFSFs. Western blotting and flow cytometry were performed to assess changes in levels of HFSF protein expression and apoptosis, respectively. Results indicated that the miR-29a mimic significantly inhibited Hsp47, Smad3, P-Smad3, and COL1A1 expression. Conversely, the miR-29a inhibitor enhanced the expression of the same genes. Furthermore, miR-29a overexpression inhibited HFSFs proliferation and enhanced the rate of HFSFs apoptosis. Consistent with this finding, miR-29a overexpression led to the downregulation of Bcl-2 and upregulation of Bax. In contrast, miR-29a suppression led to the upregulation of Bcl-2 and downregulation of Bax expression and reduced the rate of apoptosis. Additional research revealed that overexpression of Hsp47 prevented HFSFs apoptosis and enhanced collagen production. Findings that miR-29a overexpression reduces collagen expression levels, slows proliferation, and promotes apoptosis in HFSFs highlight the key role of miR-29a in scleral remodeling. The effects of miR-29a on scleral remodeling might mediate by targeting Hsp47 and repressing the Smad3 pathway.

Asiaticoside hampers epithelial-mesenchymal transition by promoting PPARG expression and suppressing P2RX7-mediated TGF-β/Smad signaling in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) accounts for 10-20% of all human ductal adenocarcinomas and has a poor prognosis relative to other subtypes because of its high propensity to develop metastases. Here, the anticancer effects of asiaticoside (AC) against TNBC and the possible underlying mechanism were examined. We found that AC inhibited the TGF-β1 expression and the SMAD2/3 phosphorylation in TNBC cells, thereby impairing the TGF-β/SMAD signaling. AC inhibited the migration, invasion, and epithelial-mesenchymal transition (EMT) of TNBC cells by suppressing the TGF-β/SMAD signaling. Meanwhile, AC inhibited the lung metastasis of TNBC cells in vivo and the expression of p-SMAD2/3 and vimentin, and increased the expression of E-cadherin and ZO-1 in the lung. Peroxisome proliferator activated receptor gamma (PPARG) was identified as a potential target of AC. AC increased PPARG expression, while PPARG knockdown attenuated the therapeutic effect of AC. AC-mediated PPARG overexpression suppressed the transcription of P2X purinoceptor 7 (P2RX7). The restoration of P2RX7 reversed the therapeutic effect of AC. These results suggested that AC blocked P2RX7-mediated TGF-β/SMAD signaling by increasing PPARG expression, thereby suppressing EMT in TNBC.

Long term toxicities following developmental exposure to perfluorooctanoic acid: Roles of peroxisome proliferation activated receptor alpha

Perfluorooctanoic acid (PFOA) is a widespread persistent organic pollutant. Fertile chicken eggs were exposed to PFOA and incubated to hatch. At three time points post hatch (0-, 1- and 3-months old), chickens were subjected to electrocardiography and sacrificed. Serum was subjected to LC-MS/MS for PFOA concentration, and organs were subjected to histopathological assessments. Additionally, PPARα-silencing lentivirus was co-applied with PFOA exposure, and the corresponding phenotypes were evaluated. Western blotting was performed to assess expressions of FABPs and pSMAD2 in heart and liver samples. Considerable amount of PFOA were detected in hatchling chicken serum, but not in one-month-old or three-month-old chicken serum. PFOA exposure resulted in developmental cardiotoxicity and hepatotoxicity in hatchling chickens. Meanwhile, one-month-old chickens still exhibited elevated heart rate, but classical cardiac remodeling (thicker right ventricular wall) were observed in exposed animals. Three-month-old chickens exhibited similar results as one-month-old ones. PPARα silencing only had partial protective effects in hatchling chickens, but the protective effects seemed to increase as chickens aged. Western blotting results indicated that L-FABP was involved in PFOA-induced hepatotoxicity, while pSMAD2 was involved in PFOA-induced cardiotoxicity. In summary, developmental exposure to PFOA resulted in persistent cardiotoxicity, but not hepatotoxicity. PPARα participates in both cardiotoxicity and hepatotoxicity.

Hesperetin modulates TGFβ induced metastatic potential of prostate cancer cells by altering histone methylation marks

Prostate cancer is the most prevalent cancer in males, and usually, death occurs due to bone metastasis. TGFβ has been shown to play an essential role in the metastasis of prostate cancer cells by promoting epithelial-to-mesenchymal transition (EMT). Hesperetin is known to possess anti-microbial, anti-fungal, antioxidant, and anti-cancer properties and good bioavailability. Therefore, this study investigated the effect of hesperetin on TGFβ-induced cell proliferation and EMT in prostate cancer cells (PC3). Interestingly, we found that hesperetin can significantly inhibit the cell proliferation of PC3 cells and arrest the cells in the S and G2M phases of the cell cycle. The invasion and migration assay results decipher the inhibitory effect of hesperetin on TGFβ-induced invasion and migration of prostate cancer cells. Our results confirmed that hesperetin also acts through the canonical signaling pathway, as we observed a significant decrease in the expression of pSmad3. Hesperetin can inhibit the TGFβ induced EMT by increasing E-cadherin expression and decreasing N-cadherin expression. Hesperetin could modulate the TGFβ induced histone methylation marks. Further investigation is required to understand the role of hesperetin in modulating these marks and thus inhibiting TGFβ-induced EMT. Hence, the results of our study identified the potential of hesperetin to modulate TGFβ-induced cell proliferation and invasion and migration of prostate cancer cells which may help inhibit the metastatic growth of prostate cancer cells.

TGF-β1 contributes to the hepatic inflammation in animal models with nonalcoholic steatohepatitis by Smad3/TLR2 signaling pathway

Non-alcoholic fatty liver disease (NAFLD) is increasingly affecting human health and the economy worldwide due to various factors. Here, we found that the expression of TGF-β1 and TLR2 was significantly up-regulated in liver samples from both rats and mice nonalcoholic steatohepatitis (NASH) models. By constructing corresponding cell model, we found that TGF-β1 challenge can positively regulate the expression of TLR2 and p-Smad2/3, and the dual luciferase reporter gene system and EMSA assay confirmed the existence of Smad3 binding site (−916 ∼ −906) in the promoter region of TLR2. The overexpression and interference changes of Smad2/3 further verified the above experimental results. Taken together, these findings suggest that TGF-β1 promotes TLR2 transcription and its target gene expression via Smad3, leading to malignant exacerbation of liver inflammation in NASH, which provides new insights into the treatment of NASH.

Myostatin is a negative regulator of adult neurogenesis after spinal cord injury in zebrafish.

Intrinsic and extrinsic inhibition of neuronal regeneration obstruct spinal cord (SC) repair in mammals. In contrast, adult zebrafish achieve functional recovery after complete SC transection. While studies of innate SC regeneration have focused on axon regrowth as a primary repair mechanism, how local adult neurogenesis affects functional recovery is unknown. Here, we uncover dynamic expression of zebrafish myostatin b (mstnb) in a niche of dorsal SC progenitors after injury. mstnb mutants show impaired functional recovery, normal glial and axonal bridging across the lesion, and an increase in the profiles of newborn neurons. Molecularly, neuron differentiation genes are upregulated, while the neural stem cell maintenance gene fgf1b is downregulated in mstnb mutants. Finally, we show that human fibroblast growth factor 1 (FGF1) treatment rescues the molecular and cellular phenotypes of mstnb mutants. These studies uncover unanticipated neurogenic functions for mstnb and establish the importance of local adult neurogenesis for innate SC repair.

Digoxin alleviates pulmonary fibrosis by regulating phosphatidylinositol-3-kinase/Akt signaling through inhibiting the activation of fibroblast: an in vivo and in vitro experiment

To investigate the effect of digoxin on bleomycin-induced pulmonary fibrosis in mice, and investigate its possible mechanism through in vitro and in vivo experiments. (1) In vivo experiment: 60 C57/BL6J mice were randomly divided into control group, pulmonary fibrosis model group (model group), pirfenidone (300 mg/kg) group, digoxin 1.0 mg/kg and 0.2 mg/kg groups, with 12 mice in each group. The pulmonary fibrosis model of mice was reproduced by single intratracheal infusion of bleomycin (5 mg/kg). The control group was given the same amount of sterile normal saline. From the next day after modeling, each group was received corresponding drugs by intragastric administration once a day for 28 days. Control group and model group were given the same amount of normal saline. The mice were sacrificed and the lung tissue was collected to detect the lung coefficient. After hematoxylin-eosin (HE) and Masson staining, the lung tissue morphology and collagen changes were observed under light microscope. Immunohistochemistry was used to detect the positive expressions of α-smooth muscle actin (α-SMA) and extracellular matrix (ECM) collagen (COL-I and COL-III) in lung tissue. The protein expressions of ECM fibronectin (FN), transforming growth factor-β (TGF-β) and phosphorylation of Smad3 (p-Smad3) in lung tissue were detected by Western blotting. (2) In vitro experiment: human embryonic lung fibroblast-1 (HFL-1) cells were cultured and divided into blank control group, fibroblast activation model group (model group), pirfenidone (2.5 mmol/L) group and digoxin 100 nmol/L and 50 nmol/L groups when cell density reached 70%-90%. After 3-hour treatment with corresponding drugs, except blank control group, the other groups were treated with TGF-β for 48 hours to establish fibroblast activation model. The expressions of α-SMA, FN and p-Smad3 proteins and the phosphorylations of phosphatidylinositol-3-kinase (PI3K)/Akt pathway proteins PI3K and Akt (p-PI3K, p-Akt) were detected by Western blotting. (1) In vivo, compared with the control group, the alveolar structure of mice in the model group was significantly damaged, a large number of inflammatory cells infiltrated, collagen deposition in the lung interstitium was increased, the deposition of ECM in the lung tissue was also increased, and the expressions of α-SMA, FN, TGF-β and p-Smad3 protein were increased, indicating that the model of bleomycin-induced pulmonary fibrosis in mice was successfully prepared. Compared with the model group, digoxin significantly inhibited airway inflammation and collagen fiber deposition, reduced ECM deposition, and decreased the protein expressions of α-SMA, FN, TGF-β and p-Smad3, while the effect was better than that of the pirfenidone group, and the digoxin 1.0 mg/kg group had a better effect except FN [α-SMA (A value): 5.37±1.10 vs. 9.51±1.66, TGF-β protein (TGF-β/GAPDH): 0.09±0.04 vs. 0.33±0.23, p-Smad3 protein (p-Smad3/GAPDH): 0.05±0.01 vs. 0.20±0.07, all P < 0.01]. (2) In vitro, compared with the blank control group, the expressions of FN, α-SMA, p-Smad3 and PI3K/Akt signaling proteins in the model group were increased, indicating that the fibroblast activation model induced by TGF-β was successfully reproduced. Compared with the model group, digoxin significantly inhibited fibroblast activation, and decreased the expressions of FN, α-SMA, p-Smad3, and PI3K/Akt pathway proteins, moreover, the effect was better than that of the pirfenidone group, and decreased FN, SMA and p-Akt protein expressions were more obvious in digoxin 100 nmol/L group [FN protein (FN/GAPDH): 0.21±0.15 vs. 0.88±0.22, α-SMA protein (α-SMA/GAPDH): 0.20±0.01 vs. 0.50±0.08, p-Akt protein (p-Akt/GAPDH): 0.30±0.01 vs. 0.65±0.10, all P < 0.01]. Digoxin could suppress the pulmonary fibrosis in mice induced by bleomycin, which might be associated with the regulation of fibroblast activation via suppressing PI3K/Akt signaling pathway in a dose-dependent manner.

Testicular orphan receptor 4 induced hepatic stellate cells activation via the regulation of TGF-β receptor Ⅰ/Smad2/3 signaling pathway

Introduction and ObjectivesLiver fibrosis is a common pathological change in many chronic liver diseases. Activation of hepatic stellate cells (HSCs) is the core event in liver fibrosis. This study aimed to investigate the role of testicular orphan receptor 4 (TR4) in the activation of HSCs. Materials and MethodsIn vivo, bile duct ligation (BDL)-induced rat liver fibrosis model was established, and the expressions of TR4 and α-smooth muscle actin (α-SMA) in liver tissues were detected. In vitro, TR4 knockdown and overexpression in JS-1 cells using lentiviral vectors were constructed, and the expressions of TR4, α-SMA, Col-I, and TGF-β1/smads and retinoid X receptor (RXR) pathway-related genes were detected. ResultsTR4 was highly expressed in BDL-induced fibrotic liver, accompanied by increased expression of α-SMA. Knockdown of TR4 significantly inhibited the expressions of α-SMA, Col-I, p-TβRI, and p-Smad2/3, and up-regulated the expression of RXRα in HSCs in vitro. In contrast, TR4 overexpression significantly increased the expressions of α-SMA, Col-I, p-TβRI, and p-Smad2/3, and inhibited the expression of RXRα. ConclusionsTR4 may promote the activation of HSCs by up-regulating TβR I/Smad2/3 signaling pathway and down-regulating RXRα signaling, thereby promoting the progression of liver fibrosis. Our findings may provide a new therapeutic target against hepatic fibrosis.

Acute toxicological study: EZY-1 with potent therapeutic effects of idiopathic pulmonary fibrosis and its mechanisms.

EZY-1 is an antifibrosis peptide purified from Eucheuma. In this study, we explored the acute toxicology of EZY-1 and the signaling pathways involved in its antifibrotic role. The mouse model of pulmonary fibrosis was induced by bleomycin. Pathological changes in lung tissue could be effectively inhibited by EZY-1. Acute toxicity and cell proliferation tests indicated that EZY-1 had no apparent toxicity to mice and cells. We identified proteins that could bind directly to EZY-1 in vitro on the basis of liquid chromatography-tandem mass spectrometry and bioinformatics analysis. EZY-1 inhibited pulmonary fibrosis via Wnt/β-catenin, transforming growth factor (TGF)-β/Smad, phosphoinositide 3-kinase/protein kinase B/ mammalian target of rapamycin, and activator of transcription 3 and Janus kinase 2/signal transducer pathways. A transwell micropore experiment showed that EZY-1 could inhibit cell migration and invasion. Western blotting analysis on transforming growth factor-β1 (TGF-β1)-induced A549 pulmonary fibrosis cell model suggested that EZY-1 could downregulate p-Smad3 (Ser423/Ser425), Smad4, β-catenin, vimentin, and N-cadherin expression. ELISA showed that EZY-1 could inhibit collagen-I secretion. EZY-1 alleviated idiopathic pulmonary fibrosis (IPF) through regulating TGF-β/Smad pathways, epithelial-mesenchymal transition processes, and collagen secretion, which provides a potential foundation for theoretical development of EZY-1 as a potential drug against IPF. PRACTICAL APPLICATIONS: We isolated a new 16-amino-acid peptide derived from the polypeptide extract of Eucheuma, named EZY-1. In vitro and in vivo assays show peptide EZY-1 is safe. The EZY-1 peptide alleviates IPF at lower doses than pirfenidone. EZY-1 alleviated idiopathic pulmonary fibrosis (IPF) through regulating TGF-β/Smad pathways, epithelial-mesenchymal transition (EMT) processes, and collagen secretion, which provides a theoretical basis for the development of EZY-1 as a potential drug against IPF.

Upregulation of miR-222-3p alleviates the symptom of aortic dissection through targeting STAT3

ObjectiveThis study sought to investigate the differentially expressed miRNAs in Aortic dissection (AD) and explore the downstream mechanisms in regulating AD. MethodsExosomes of AD patients and healthy people were isolated by differential centrifugation, and the differentially expressed miRNAs were evaluated by RNA sequencing. The downstream target of miR-222-3p was predicted by bioinformatics method and validated by dual-luciferase assay. Angiotensin II and Promethazine were used to establish AD mouse model and platelet-derived growth factor BB (PDGF-BB) was used to induce human vascular smooth muscle cells (HVSMCs) to elucidate the effect of miR-222-3p upregulation on AD in vivo and in vitro. The relative level of miR-222-3p was evaluated by RT-qPCR. The level of several proteins was investigated by Western blot. Immunofluorescence staining was used to detect the stress fiber formation. Cell migration was evaluated by wound healing and Transwell assay. The proliferation, cell cycle and apoptosis of HVSMCs were assessed by CCK-8 and flow cytometry, respectively. ResultsMiR-222-3p was downregulated in AD and PDGF-BB induced HVSMCs. The upregulation of miR-222-3p alleviated the symptom of AD in vivo by targeting STAT3, and inhibited stress fiber formation, abnormal migration, proliferation and apoptosis of HVSMCs induced by PDGF-BB by regulating the expression of α-SMA, SM22α, MMP2, MMP9 and p-Smad2. ConclusionThe upregulation of miR-222-3p attenuates the progression of AD. Our study provides a theoretical basis for exploring new strategies against AD.

Preventive Effect and Mechanism of Anthocyanins from Aronia Melanocarpa Elliot on Hepatic Fibrosis Through TGF-β/Smad Signaling Pathway.

In order to explore the effect and mechanism of Aornia mealnocarpa Elliot anthocyanins (AMA) at the cellular level on hepatic fibrosis (HF), molecular docking, RT-PCR and Western Blotting were used to explore the molecular mechanism and the effects of different doses AMA on HSC-T6 cells by TGF-β1 induction. The results showed that the binding energy of anthocyanins on TGF-β1 (PDB ID: 3KFD) was in the range of -9.5 to 8.6 kcal/mol, with good low energy parameters and binding positions. AMA could effectively inhibit the expressions of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and total serum bilirubin (TSB), and improved the expressions of total protein (TP) and albumin (ALB). RT-PCR and Western bloting results showed that AMA could inhibited the secretion of inflammatory cytokines IL-1, IL-6, TNF-α and COX-2, and inhibit the expression of TGF-β1, P-Smad2, α-SMA and Collagen I in TGF-β /Smad signaling pathway. This study revealed the AMA's inhibition effects and mechanism of malignant biological behavior of HSC-T6 cells, in order to provide theoretical basis for the prevention and treatment of HF by Aronia melanocarpa Elliot.

Dysregulated myokines and signaling pathways in skeletal muscle dysfunction in a cigarette smoke-induced model of chronic obstructive pulmonary disease.

Skeletal muscle dysfunction is an important extrapulmonary comorbidity of chronic obstructive pulmonary disease (COPD). Muscle-derived cytokines (myokines) play important roles in skeletal muscle growth and function, but their contributions to skeletal muscle dysfunction in COPD have not been fully understood. In the current study, by using a well-established mouse model of COPD with skeletal muscle dysfunction, we found that the expressions of Fndc5 (fibronectin type III domain-containing protein 5, the precursor of irisin) and peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) were decreased, while myostatin (Mstn), phosphorylated extracellular regulated kinase (p-Erk1/2), and p-Smad3 expressions were upregulated in skeletal muscles from cigarette smoke-exposed mice and in cigarette smoke extract (CSE)-stimulated C2C12 myotubes. Treatment with Smad3 or Erk1/2 inhibitors partially restored the expression of Fndc5 in CSE-stimulated C2C12 myotubes. Taken together, CSE exposure, by upregulation of p-Erk1/2, promoted the expression of Mstn, which further inhibited Fndc5 expression by the p-Smad3/PGC-1α pathway, revealing a novel regulating mechanism of myokines in the pathogenesis of skeletal muscle comorbidities of COPD.