Pharmacological inhibition of myostatin/TGF-β receptor/pSmad3 signaling rescues muscle regenerative responses in mouse model of type 1 diabetes

Aim:

To study the influence of acute experimental diabetes on the regenerative potential of muscle stem (satellite) cells in mice.

Methods:

Male C57BL/6 young mice were injected with a single dose of streptozotocin (STZ, 180 mg/kg, ip) to induce diabetes. The diabetic mice were treated with insulin (0.75 U/kg, ip), follistatin (12 μg/kg, im) or Alk5 inhibitor (5 μmol/L per kg, sc) once a day. On the first day when high glucose levels were found, cardiotoxin (CTX) was focally injected into tibialis anterior and gastronemius muscles of the mice. The muscles were harvested 3 d and 5 d after CTX injection, and myofibers and satellite cells were isolated. Quantitative ex-vivo and in-vivo assays of myogenic potential were used to evaluate the muscle regenerative responses.

Results:

The satellite cells from the diabetic mice 3 d after CTX injection fail to activate, and the repair of muscle deteriorates, resembling that observed in old control mice. Furthermore, the satellite cells have excessive levels of myostatin, TGF-β receptor 1, pSmad3 and the cell cycle inhibitor p15, while the level of TGF-β1 remain unchanged. Treatment of the diabetic mice with insulin rescued muscle regenerative responses, and restored the expression levels of myostatin, TGF-β receptor 1, pSmad3, and p15 to those similar of healthy controls. Treatment of the diabetic mice with the myostatin antagonist follistatin, or with the Alk5 inhibitor of TGF-β receptor 1 (which did not diminish the blood glucose levels) rescued muscle regenerative responses and attenuated the myostatin/TGFβ receptor/pSmad3 signaling.

Conclusion:

The muscle regenerative responses are incapacitated and repair of the tissue fails within hours after the initiation of hyperglycemia in a mouse model of type 1 diabetes, but stem cell function is rescued by insulin, as well as follistatin or an Alk5 inhibitor that blocks TGF-β receptor signaling.     

The anti-fibrotic effects of epigallocatechin-3-gallate in bile duct-ligated cholestatic rats and human hepatic stellate LX-2 cells are mediated by the PI3K/Akt/Smad pathway

Aim:

(−)-Epigallocatechin-3-gallate (EGCG) is one of the most abundant polyphenols in green tea with strong antioxidant activity and various therapeutic effects. In this study, we investigated the anti-fibrotic effects of EGCG and underlying mechanisms in bile duct-ligated (BDL) rats and a liver fibrosis model in vitro.

Methods:

BDL rats were treated with EGCG (25 mg·kg⁻¹·d⁻¹, po) for 14 d, and then the serum, bile and liver samples were collected. Liver fibrosis was assessed by serum, urine and bile biochemistry analyses and morphological studies of liver tissues. TGF-β1-stimulated human hepatic stellate LX-2 cells were used as a liver fibrosis model in vitro. The expression of liver fibrogenic genes and signaling proteins in the PI3K/Akt/Smad pathway was examined using Western blotting and/or real-time PCR.

Results:

In BDL rats, EGCG treatment significantly ameliorates liver necrosis, inflammation and fibrosis, and suppressed expression of the genes associated with liver inflammation and fibrogenesis, including TNF-α, IL-1β, TGF-β1, MMP-9, α-SMA, and COL1A1. In LX-2 cells, application of EGCG (10, 25 μmol/L) dose-dependently suppressed TGF-β1-stimulated expression of COL1A1, MMP-2, MMP-9, TGF-β1, TIMP1, and α-SMA. Furthermore, EGCG significantly suppressed the phosphorylation of Smad2/3 and Akt in the livers of BDL rats and in TGF-β1-stimulated LX-2 cells. Application of {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, a specific inhibitor of PI3K, produced similar effects as EGCG did in TGF-β1-stimulated LX-2 cells, but co-application of EGCG and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 did not produce additive effects.

Conclusion:

EGCG exerts anti-fibrotic effects in BDL rats and TGF-β1-stimulated LX-2 cells in vitro via inhibiting the PI3K/Akt/Smad pathway.     

Induction of cell-proliferation hormesis and cell-survival adaptive response in mouse hematopoietic cells by whole-body low-dose radiation.

Hormesis and a cytogenetic adaptive response induced by low-dose radiation (LDR) have been extensively documented. However, few studies have investigated the induction of an adaptive response by LDR for cell survival in vitro. In the present study, we investigated whether LDR could induce hormesis in hematopoietic cells and the adaptive response of these cells to subsequent high-dose radiation-induced cytotoxic effects. Mice were exposed in whole-body to 0 (as control), 0.05, 0.25, 0.50, 0.75, and 1.00 Gy of X-rays. They were killed 12, 24, 48, and 72 h later to observe the stimulating effect of LDR on total bone marrow cells per femur and bone marrow progenitor, colony-forming unit-granulocyte-macrophage (CFU-GM). Exposure to 0.5 Gy of X-rays resulted in significantly stimulating effects on both parameters with a maximum effect at 48 h, showing a cell-proliferation hormesis. In the next experiment, mice were irradiated by 0.5 Gy X-rays as an adaptive exposure (D1), and 6, 12, 24, 48, and 72 h later, they were exposed to 6 Gy X-rays as a challenging exposure (D2). Forty-eight h after D2, cytotoxic effects were analyzed using peripheral blood cells (red blood cells, white blood cells, and platelets) and bone marrow cells (total bone marrow cells of the femur, and bone marrow progenitors such as CFU-GM and erythroid burst-forming unit, BFU-E). An adaptive response to D2-induced cytotoxic effect, named as the cell-survival adaptive response, was found in both peripheral blood cells and bone marrow cells when D1 and D2 exposures were given at intervals of 24-48 h. These results suggested that LDR could induce both cell-proliferation hormesis and cell-survival adaptive response to subsequent high-dose radiation in bone marrow cells. It may be of potential importance, if this phenomenon is confirmed clinically, since it may be applied to reduce the adverse effect of radiotherapy.     

Effect of aqueous extract of Achillea millefolium on the development of experimental autoimmune encephalomyelitis in C57BL/6 mice

Objective:

Achillea millefolium (A. millefolium) is widely used as an anti-inflammatory remedy in traditional and herbal medicine. In this study, we investigated the effect of an aqueous extract from A. millefolium on experimental autoimmune encephalomyelitis (EAE) and on the serum cytokine levels in C57BL/6 mice.

Materials and Methods:

EAE was induced in 63 C57BL/6 mice weighing 20-25 g (8 weeks old). Following immunization, the treatment protocol was initiated by using different doses of an aqueous extract from A. millefolium (1, 5, and 10 mg/mouse/day). Histopathologic assessments were performed by hematoxylin and eosin (H and E) and luxol fast blue (LFB) staining. Behavioral disabilities were recorded by a camera. Serum levels of interleukin (IL)-10, IL-12, and transforming growth factor (TGF)-β were measured using enzyme-linked immunosorbent assay (ELISA).

Results:

On average, mice developed classical behavioral disabilities of EAE, 13.2 ± 1.9 days following immunization. Treatment of mice with A. millefolium led to delay the appearance of behavioral disabilities along with reduced severity of the behavioral disabilities. Treatment with A. millefolium prevented weight loss and increased serum levels of TGF-β in immunized mice with MOG35-55. EAE-induced mice, which were treated with A. millefolium, had less cerebral infiltration of inflammatory cells.

Conclusion:

The results demonstrated that treatment with aqueous extract of A. millefolium may attenuate disease severity, inflammatory responses, and demyelinating lesions in EAE-induced mice. In addition, following treatment with A. millefolium, serum levels of TGF-βwere increased in EAE-induced mice.KEY WORDS: Achillea millefolium, cytokines, experimental autoimmune encephalomyelitis, multiple sclerosis     

Ursodeoxycholyl lysophosphatidylethanolamide attenuates hepatofibrogenesis by impairment of TGF-β1/Smad2/3 signalling

Background and Purpose

Chronic hepatic inflammation results in liver fibrosis. As effective anti-fibrogenic agents are lacking, we investigated ursodeoxycholyl lysophosphatidylethanolamide (UDCA-LPE), a synthetic bile acid–phospholipid conjugate with anti-inflammatory and anti-apoptotic properties for tis effects on hepatic fibrogenesis.

Experimental Approach

To stimulate fibrogenesis, LX2 hepatic stellate cells were cultured with conditioned medium from CL48 liver cells after exposure to stress-inducing conditions – methionine–choline-deficient (MCD) medium or TNFα/cycloheximide (CHX) – with or without UDCA-LPE preincubation. Anti-fibrogenic effects of UDCA-LPE were further studied in CL48 and LX2 cells and in primary human hepatic stellate cells (HHStec) directly exposed to TGF-β1. To test UDCA-LPE in vivo, C57BL/6 mice were fed a MCD diet for 11 weeks followed by 30 mg·kg⁻¹ UDCA-LPE 3× per week for 2.5 weeks.

Key Results

Expression of α-smooth muscle actin (α-SMA), α1-collagen, vimentin and TGF-β1 was down-regulated by up to 93% by UDCA-LPE in LX-2 cells cultured with conditioned medium. Also, UDCA-LPE inhibited Smad3 phosphorylation in CL48 cells incubated with MCD medium or TNFα/CHX and in LX2 cells exposed to conditioned medium. UDCA-LPE also decreased phosphorylated Smad3 and Smad2 directly induced by TGF-β1. Inhibition of TGF-β1/Smad2/3 signalling with down-regulation of target genes was confirmed in HHStec. In vivo, UDCA-LPE decreased hepatic α-SMA, α1-collagen and TGF-β1 expression and markedly lowered α-SMA protein and collagen deposition in MCD mice.

Conclusions and Implications

By blocking TGF-β1/Smad2/3 signalling, UDCA-LPE suppressed key mediators of hepatic fibrogenesis. Thus, UDCA-LPE could be suitable for prevention of fibrotic progression of chronic liver disease.     

Modification of gamma radiation induced response of peritoneal macrophages and splenocytes by Hippophae rhamnoides (RH-3) in mice

Alcoholic extract of Hippophae rhamnoides, RH-3, reported to render >80% survival against lethal whole body Co-60-gamma irradiation (10 Gy) in mice, was investigated for its immunostimulatory effects. In comparison with un-irradiated control, whole body irradiation did not reduce peritoneal macrophage counts at 24 h post-irradiation. RH-3 treatment (30 mg kg(-1) body weight) alone or 30 min before whole-body irradiation enhanced viable counts of macrophages significantly (P< or =0.05) compared with both un-irradiated control and irradiated groups. Whole-body irradiation reduced the number of viable splenocytes significantly (P<0.05) compared with un-irradiated control at 24 h post-irradiation. RH-3 treatment alone or before whole-body irradiation appreciably countered radiation-induced decrease in splenocyte count. 3H-thymidine uptake method revealed that whole-body irradiation reduced splenocyte proliferation significantly (159 +/- 45 counts min(-1)/10(6) cells; P< or =0.05) in comparison with control (607 +/- 142 counts min(-1)) at 24 h after irradiation but RH3 treatment before irradiation reduced the steep decrease and maintained it as 444+/-153 counts min(-1). After whole-body irradiation, the ratio of spleen weight/mouse weight decreased to 1.5 +/- 04 compared with 2.9 +/- 0.32 in un-irradiated control at 24 h post-irradiation. Similarly, total protein content in splenocytes also decreased to 48 +/- 6 microg/10(6) cells in comparison with 368 +/- 16 microg/10(6) cells of un-irradiated control. RH-3 treatment before irradiation countered radiation-induced decrease in both spleen weight/mouse weight ratio (4.0 +/- 0.35) and total protein content (360 +/- 13 mug/10(6) splenocytes). In the supernatant of peritoneal macrophage cultures exposed to 2 Gy Co-60-gamma radiation ex-vivo, the total nitrite content was enhanced significantly (P<0.05) to 5.72 +/- 0.09 microM in comparison with un-irradiated control (1.64 +/- 0.09 microM). RH-3 treatment (30 microg mL(-1)) before irradiation reduced total nitrite significantly (0.93 +/- 0.3; P< or =0.05) in comparison with irradiated control group. At 24 h after whole body irradiation, the CD4+/CD8+ ratio reduced to 1.5 in comparison with un-irradiated control (1.9) but RH-3 treatment before irradiation restored the ratio to 2.1. These findings explicitly reveal the immunostimulatory activity of RH-3, which may play an important role in the manifestation of its radioprotective efficacy.     

Over-production of nitric oxide by oxidative stress-induced activation of the TGF-β1/PI3K/Akt pathway in mesangial cells cultured in high glucose

Aim:

To investigate whether NO over-production in rat mesangial cells cultured in high glucose (HG) is related to activation of the TGF-β1/PI3K/Akt pathway.

Methods:

Rat mesangial cells line (HBZY-1) was exposed to HG (24.44 mmol/L) or H₂O₂ (10 μmol/L) for 16 h. NO release was quantified using the Griess assay. The TGF-β1 level was measured using ELISA. The protein expression of p-Akt, t-Akt, Bim, and iNOS was examined by Western blotting. The mRNA levels of TGF-β1 and Bim were measured using RT-PCR. The cell proliferation rate was estimated using a BrdU incorporation assay.

Results:

Treatment of the cells with HG, H₂O₂, or TGF-β1 (5 ng/mL) significantly increased the NO level that was substantially inhibited by co-treatment with the NADPH oxidase inhibitor diphenylene iodonium (DPI), TGF-β1 inhibitor SB431542, or PI3K inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002. Both HG and H₂O₂ significantly increased the protein and mRNA levels of TGF-β1 in the cells, and HG-induced increases of TGF-β1 protein and mRNA were blocked by co-treatment with DPI. Furthermore, the treatment with HG or H₂O₂ significantly increased the expression of phosphorylated Akt and iNOS and cell proliferation rate, which was blocked by co-treatment with DPI, SB431542, or {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002. Moreover, the treatment with HG or H₂O₂ significantly inhibited Bim protein and mRNA expression, which was reversed by co-treatment with DPI, SB431542, or {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002.

Conclusion:

The results demonstrate that high glucose causes oxidative stress and NO over-production in rat mesangial cells in vitro via decreasing Bim and increasing iNOS, which are at least partially mediated by the TGF-β1/PI3K/Akt pathway.     

Moxifloxacin modifies corneal fibroblast-to-myofibroblast differentiation

Background and Purpose

Fibroblast-to-myofibroblast differentiation is associated with scarring, an important issue in corneal surgery. Moxifloxacin (MOX), commonly applied to prevent post-operative infection, would benefit more if it modifies fibroblast-to-myofibroblast differentiation other than antimicrobial activity. Our purpose was to explore whether MOX has anti-fibrotic effect in human corneal fibroblasts (HCFs).

Experimental Approach

HCFs were incubated in MOX-containing medium concurrently with TGF-β1 (co-treatment), before (pretreatment) or after (post-treatment) adding TGF-β1. HCF contractility was evaluated with a type I collagen gel contraction assay. Expression of α-smooth muscle actin (α-SMA), Smad2, phospho-Smad2-Ser467, Smad4 and Smad7 was determined by immunoblotting. Formation of α-SMA-positive filaments and distribution of active Smad2 were observed under confocal microscopy. Expression of TGF-β receptor types I (TGFBR1) and II (TGFBR2) was assessed with flow cytometry.

Key Results

MOX did not affect gel contractility or α-SMA filament formation in HCFs without TGF-β1 stimulation. MOX did, however, retard HCF-containing gel contractility and α-SMA filament formation following TGF-β1 stimulation in the pretreatment and co-treatment groups but not in the post-treatment group. MOX blocked the expression of Smad2, phospho-Smad2-Ser467 and TGFBR1 under TGF-β1 incubation. Additionally, MOX enhanced Smad7 expression in TGF-β1-incubated HCFs, but did not interfere with TGF-β-triggered Smad2 nuclear translocation or Smad4 expression.

Conclusions and Implications

MOX inhibited TGF-β-induced fibroblast-to-myofibroblast differentiation via blocking TGFBR1 and enhancing Smad7 expression. MOX should be used before or during surgery to achieve these effects. These results suggest a de novo mechanism by which MOX participates in corneal wound healing.     

Evaluation of the radioprotective effects of propolis and flavonoids in gamma-irradiated mice: the alkaline comet assay study

The radioprotective effects of water-soluble derivate of propolis (WSDP) collected in Croatia, and single flavonoids, caffeic acid, chrysin and naringin in the whole-body irradiated CBA mice were investigated. Irradiation was performed using a gamma-ray source ((60)Co), and absorbed doses were 4 and 9 Gy. The efficiency of test components was evaluated when given intraperitoneally (i.p.) at dose of 100 mg kg(-1) for 3 consecutive days before and/or after irradiation. Moreover, possible genotoxic effects of all test components were assessed on non-irradiated animals. The higher efficiency of test components was observed when given preventively. The results suggest that propolis and related flavonoids given to mice before irradiation protected mice from lethal effects of whole-body irradiation and diminish primary DNA damage in their white blood cells as detected by the alkaline comet assay.     

MS80, a novel sulfated oligosaccharide,inhibits pulmonary fibrosis by targeting TGF-β1 both in vitro and in vivo

Aim:

The pro-fibrogenic cytokine transforming growth factor-beta 1 (TGF-β1) has attracted much attention for its potential role in the etiology of idiopathic pulmonary fibrosis (IPF). Here, we demonstrate that MS80, a novel sulfated oligosaccharide extracted from seaweed, can bind TGF-β1. The aim of the present study was to determine whether MS80 is capable of combating TGF-β1-mediated pulmonary fibrotic events both in vitro and in vivo, and to investigate the possible underlying mechanisms.

Methods:

Surface plasmon resonance was used to uncover the binding profiles between the compound and TGF-β. MTT assay, flow cytometry, Western blot analysis, BCA protein assay and SDS-PAGE gelatin zymography were used to probe the antifibrotic mechanisms of MS80. The in vivo fibrotic efficacy was evaluated in a bleomycin instillation-induced rat model.

Results:

We report that MS80, a new kind of sulfated oligosaccharide extracted from seaweed, inhibits TGF-β1-induced pulmonary fibrosis in vitro and bleomycin-induced pulmonary fibrosis in vivo. Our results indicated that MS80 competitively inhibited heparin/HS-TGF-β1 interaction through its high binding affinity for TGF-β1. Moreover, MS80 arrested TGF-β1-induced human embryo pulmonary fibroblast (HEPF) cell proliferation, collagen deposition and matrix metalloproteinase (MMP) activity. Intriguingly, MS80 deactivated both the ERK and p38 signaling pathways. MS80 was also a potent suppressor of bleomycin-induced rat pulmonary fibrosis in vivo, as evidenced by improved pathological settings and decreased lung collagen contents.

Conclusion:

MS80 in particular, and perhaps oligosaccharide in general, offer better pharmacological profiles with appreciably few side effects and represent a promising class of drug candidates for IPF therapy.     

Acute and chronic administration of SHR117887, a novel and specific dipeptidyl peptidase-4 inhibitor,improves metabolic control in diabetic rodent models

Aim:

Dipeptidyl deptidase-4 (DPP-4) inhibitors are a new class of anti-diabetic agents. The purpose of this study was to assess the acute and chronic effects of SHR117887, a novel DPP-4 inhibitor, on metabolic control and pancreatic β-cell function in normal or diabetic rodent models.

Methods:

In the acute experiments, ICR mice, diet-induced obese (DIO) rats and ob/ob mice were subjected to an oral glucose tolerance test (OGTT) following a single oral administration of SHR117887 (0.1, 0.3, 1 or 3 mg/kg). Blood samples were collected to measure glucose, insulin, DPP-4 activity and active GLP-1 level. In the chronic experiments, ob/ob mice was administered SHR117887 (3, 10 or 30 mg/kg) twice daily for 33 d to assess the effects on metabolic control and pancreatic β-cell function. Vildagliptin (LAF237) was used as a positive control in all the experiments.

Results:

Acute oral administration of SHR117887 dose-dependently decreased the serum DPP-4 activity and improved glucose tolerance in ICR mice, DIO rats and ob/ob mice. This was accompanied by significant increases in the serum active GLP-1 and insulin levels. Chronic administration of SHR117887 significantly decreased fasting blood glucose level and improved the lipid profiles in ob/ob mice by reducing the serum triglyceride and free fatty acid levels, and its efficacy was comparable with that of vildagliptin at the same molarity. Moreover, chronic administration of SHR117887 increased the insulin staining of islet cells, which is suggestive of improved β-cell function.

Conclusion:

SHR117887 is a potent DPP-4 inhibitor that improves metabolic control and β-cell function in diabetic rodent models, suggesting that it could be a new therapeutic agent for the treatment of type 2 diabetes.     

Co-delivery of ccl19 gene enhances anti-caries DNA vaccine pCIA-P immunogenicity in mice by increasing dendritic cell migration to secondary lymphoid tissues

Aim:

To investigate how co-delivery of the gene encoding C–C chemokine ligand-19 (CCL-19) affected the systemic immune responses to an anti-caries DNA vaccine pCIA-P in mice.

Methods:

Plasmid encoding CCL19-GFP fusion protein (pCCL19/GFP) was constructed by inserting murine ccl19 gene into GFP-expressing vector pAcGFP1-N1. Chemotactic effect of the fusion protein on murine dendritic cells (DCs) was assessed in vitro and in vivo using transwell and flow cytometric analysis, respectively. BALB/c mice were administered anti-caries DNA vaccine pCIA-P plus pCCL19/GFP (each 100 μg, im) or pCIA-P alone. Serum level of anti-PAc IgG was assessed with ELISA. Splenocytes from the mice were stimulated with PAc protein for 48 h, and IFN-γ and IL-4 production was measured with ELISA. The presence of pCCL19/GFP in spleen and draining lymph nodes was assessed using PCR. The expression of pCCL19/GFP protein in these tissues was analyzed under microscope and with flow cytometry.

Results:

The expression level of CCL19-GFP fusion protein was considerably increased 48 h after transfection of COS-7 cells with pCCL19/GFP plasmids. The fusion protein showed potent chemotactic activity on DCs in vitro. The level of serum PAc-specific IgG was significantly increased from 4 to 14 weeks in the mice vaccinated with pCIA-P plus pCCL19/GFP. Compared to mice vaccinated with pCIA-P alone, the splenocytes from mice vaccinated with pCIA-P plus pCCL19/GFP produced significantly higher level of IFN-γ, but IL-4 production had no significant change. Following intromuscular co-delivery, pCCL19/GFP plasmid and fusion protein were detected in the spleen and draining lymph nodes. Administration of CCL19 gene in mice markedly increased the number of mature DCs in secondary lymphoid tissues.

Conclusion:

CCL19 serves as an effective adjuvant for anti-caries DNA vaccine by inducing chemotactic migration of DCs to secondary lymphoid tissues.     

Dose and dose rate effects of whole-body gamma-irradiation: I. Lymphocytes and lymphoid organs

The major goal of part I of this study was to compare varying doses and dose rates of whole-body gamma-radiation on lymphoid cells and organs. C57BL/6 mice (n = 75) were exposed to 0, 0.5, 1.5, and 3.0 Gy gamma-rays (60Co) at 1 cGy/min (low-dose rate, LDR) and 80 cGy/min (high-dose rate, HDR) and euthanized 4 days later. A significant dose-dependent loss of spleen mass was observed with both LDR and HDR irradiation; for the thymus this was true only with HDR. Decreasing leukocyte and lymphocyte numbers occurred with increasing dose in blood and spleen at both dose rates. The numbers (not percentages) of CD3+ T lymphocytes decreased in the blood in a dose-dependent manner at both HDR and LDR. Splenic T cell counts decreased with dose only in HDR groups; percentages increased with dose at both dose rates. Dose-dependent decreases occurred in CD4+ T helper and CD8+ T cytotoxic cell counts at HDR and LDR. In the blood the percentages of CD4+ cells increased with increasing dose at both dose rates, whereas in the spleen the counts decreased only in the HDR groups. The percentages of the CD8+ population remained stable in both blood and spleen. CD19+ B cell counts and percentages in both compartments declined markedly with increasing HDR and LDR radiation. NK1.1+ natural killer cell numbers and proportions remained relatively stable. Overall, these data indicate that the observed changes were highly dependent on the dose, but not dose rate, and that cells in the spleen are more affected by dose rate than those in blood. The results also suggest that the response of lymphocytes in different body compartments may be variable.     

Salvianolic acid B attenuates tubulointerstitial fibrosis by inhibiting EZH2 to regulate the PTEN/Akt pathway

ContextSalvianolic acid B (SAB) can alleviate renal fibrosis and improve the renal function.ObjectiveTo investigate the effect of SAB on renal tubulointerstitial fibrosis and explore its underlying mechanisms.Materials and methodsMale C57 mice were subjected to unilateral ureteric obstruction (UUO) and aristolochic acid nephropathy (AAN) for renal fibrosis indication. Vehicle or SAB (10 mg/kg/d, i.p.) were given consecutively for 2 weeks in UUO mice while 4 weeks in AAN mice. The serum creatinine (Scr) and blood urine nitrogen (BUN) were measured. Masson’s trichrome staining and the fibrotic markers (FN and α-SMA) were used to evaluate renal fibrosis. NRK-49F cells exposed to 2.5 ng/mL TGF-β were treated with SAB in the presence or absence of 20 μM 3-DZNep, an inhibitor of EZH2. The protein expression of EZH2, H3k27me3 and PTEN/Akt signaling pathway in renal tissue and NRK-49F cells were measured by Western blots.ResultsSAB significantly improved the levels of Scr by 24.3% and BUN by 35.7% in AAN mice. SAB reduced renal interstitial collagen deposition by 34.7% in UUO mice and 72.8% in AAN mice. Both in vivo and in vitro studies demonstrated that SAB suppressed the expression of FN and α-SMA, increased PTEN and decreased the phosphorylation of Akt, which were correlated with the down-regulation of EZH2 and H3k27me3. The inhibition of EZH2 attenuated the anti-fibrotic effects of SAB in NRK-49Fs.ConclusionSAB might have therapeutic potential on renal fibrosis of CKD through inhibiting EZH2, which encourages further clinical trials.     

不同剂量137Csγ-射线辐射后造血干/祖细胞辐射敏感性差异研究

摘要：目的 探讨不同剂量137Csγ-射线照射后不同时间点C57 BL/6小鼠造血细胞放射敏感性的差异。方法 选取6~8周龄雄性C57BL/6小鼠72只,完全随机法分为对照组和照射组。照射组小鼠分别接受2、4、6 Gy137Csγ射线一次性全身照射,对照组小鼠接受假照射。小鼠分别于受照后14 d、35d和56d断颈处死,取外周血进行血象测定,取骨髓细胞测定有核细胞数目和造血干/祖细胞数目。结果 不同剂量照射后小鼠的外周血常规指标有明显变化,白细胞数目明显下降,其次是血小板数目,且具有剂量效应关系;在照射后14d,2Gy、4Gy和6Gy照射组小鼠骨髓有核细胞数目与对照组比较分别下降21.9%、39.9%和54.4%（t=4.311、6.401、8.007,P<0.05）;照射后35d和56d,6Gy照射组小鼠骨髓有核细胞和造血祖细胞数目显著低于对照组（t=4.185、3.596,P<0.05）。照射后14d、35d和56d,2Gy、4Gy和6Gy照射组小鼠骨髓造血干细胞数目持续低于对照组（t=9.706、3.427~7.465,P<0.05）。结论 不同剂量137Csγ-射线照射对小鼠造血系统造成不同程度的损伤,造血祖细胞较造血干细胞辐射敏感,且辐射对造血干细胞造成的损伤是持久性的。     

An in vitro model for the pro-fibrotic effects of retinoids: mechanisms of action

BACKGROUND AND PURPOSE

Retinoids, including all-trans retinoic acid (tRA), have dose-dependent pro-fibrotic effects in experimental kidney diseases. To understand and eventually prevent such adverse effects, it is important to establish relevant in vitro models and unravel their mechanisms.

EXPERIMENTAL APPROACH

Fibrogenic effects of retinoids were assessed in NRK-49F renal fibroblasts using picro-Sirius red staining for collagens and quantified by spectrophotometric analysis of the eluted stain. Other methods included RT-qPCR, immunoassays and matrix metalloproteinase (MMP) activity assays.

KEY RESULTS

With or without TGF-β1, tRA was dose-dependently pro-fibrotic, notably increasing collagen accumulation. tRA and TGF-β1 additively suppressed expression of mRNA for MMP2, 3 and 13 and suppressed MMP activity. tRA, in the presence of TGF-β1, induced plasminogen activator inhibitor-1 (PAI-1) mRNA and they additively induced PAI-1 protein expression. A PAI-1 inhibitor, a pan-retinoic acid receptor (RAR) antagonist and a pan-retinoid X receptor (RXR) antagonist each partially prevented the pro-fibrotic effect of tRA. The dose-dependent pro-fibrotic effects of a pan-RXR agonist were similar to those of tRA. A pan-RAR agonist showed weaker, less dose-dependent pro-fibrotic effects and the pro-fibrotic effects of RARα and RARβ-selective agonists were even smaller. An RARγ-selective agonist did not affect fibrogenesis.

CONCLUSIONS AND IMPLICATIONS

An in vitro model for the pro-fibrotic effects of retinoids was established in NRK-49F cells. It was associated with reduced MMP activity and increased PAI-1 expression, and was probably mediated by RXR and RAR. To avoid or antagonize the pro-fibrotic activity of tRA, further studies on RAR isotype-selective agonists and PAI-1 inhibitors might be of value.     

PTPL1 suppresses lung cancer cell migration via inhibiting TGF-β1-induced activation of p38 MAPK and Smad 2/3 pathways and EMT

Epithelial-mesenchymal transition (EMT) enables dissemination of neoplastic cells and onset of distal metastasis of primary tumors. However, the regulatory mechanisms of EMT by microenvironmental factors such as transforming growth factor-β (TGF-β) remain largely unresolved. Protein tyrosine phosphatase L1 (PTPL1) is a non-receptor protein tyrosine phosphatase that plays a suppressive role in tumorigenesis of diverse tissues. In this study we investigated the role of PTPL1/PTPN13 in metastasis of lung cancer and the signaling pathways regulated by PTPL1 in terms of EMT of non-small cell lung cancer (NSCLC) cells. We showed that the expression of PTPL1 was significantly downregulated in cancerous tissues of 23 patients with NSCLC compared with adjacent normal tissues. PTPL1 expression was positively correlated with overall survival of NSCLC patients. Then we treated A549 cells in vitro with TGF-β1 (10 ng/mL) and assessed EMT. We found that knockdown of PTPL1 enhanced the migration and invasion capabilities of A549 cells, through enhancing TGF-β1-induced EMT. In nude mice bearing A549 cell xenografts, knockdown of PTPL1 significantly promoted homing of cells and formation of tumor loci in the lungs. We further revealed that PTPL1 suppressed TGF-β-induced EMT by counteracting the activation of canonical Smad2/3 and non-canonical p38 MAPK signaling pathways. Using immunoprecipitation assay we demonstrated that PTPL1 could bind to p38 MAPK, suggesting that p38 MAPK might be a direct substrate of PTPL1. In conclusion, these results unravel novel mechanisms underlying the regulation of TGF-β signaling pathway, and have implications for prognostic assessment and targeted therapy of metastatic lung cancer.