γ-分泌酶抑制剂在肺纤维化上皮间质转化中的作用

目的 检测体外转化生长因子（TGF）-β1诱导肺纤维化上皮间质转化（EMT）的形成情况并检测Notch信号特异性抑制剂γ-分泌酶抑制剂（DAPT）对其影响及可能机制。方法 将A549细胞分成对照组（RPMI 1640完全培养基培养）、TGF-β1组（在含有10 μg/L TGF-β1的RPMI 1640培养基中培养）、TGF-β1+DAPT组（在含有10 μg/L TGF-β1和2 μmol DAPT的RPMI 1640培养基中培养）、DAPT组（在含有2 μmol DAPT的RPMI 1640培养基中培养）。通过倒置显微镜观察细胞形态,实时荧光定量逆转录聚合酶链反应检测肺泡上皮细胞特异性蛋白E-钙黏合素以及间质细胞特异性蛋白α-肌动蛋白（α-SMA）mRNA相对表达水平,Western blot检测E-钙黏合素以及α-SMA蛋白表达水平。结果 倒置显微镜检查示,对照组细胞呈多边形,铺路石样,细胞间连接紧密;TGF-β1组细胞呈梭行,纺锤状,细胞间连接减少;TGF-β1+DAPT组仅有少量细胞呈梭形,多数细胞形态与对照组相似;DAPT组细胞形态大致同对照组。与对照组相比,TGF-β1组α-SMA蛋白及mRNA表达增加,而E-钙黏合素蛋白及mRNA表达减弱（P<0.05）;与TGF-β1组比较,TGF-β1+DAPT组α-SMA蛋白及mRNA表达水平降低,而E-钙黏合素蛋白及mRNA表达水平增加（P<0.05）;DAPT组与对照组2指标的蛋白与mRNA相对表达水平差异无统计学意义（P>0.05）。结论 TGF-β1可诱导肺上皮间质转化,而Notch信号抑制剂DAPT能够阻断、部分或全部逆转这一过程。     

VEGF ameliorates tubulointerstitial fibrosis in unilateral ureteral obstruction mice via inhibition of epithelial-mesenchymal transition

Aim:

Vascular endothelial growth factor (VEGF) has been shown to be a survival factor for renal tubular epithelial cells. In the present study, we investigated whether administration of VEGF ameliorates tubulointerstitial fibrosis in a mouse model of unilateral ureteral obstruction (UUO).

Methods:

Thirty-six male CD-1 mice were randomly divided into three groups: sham-operation, UUO and UUO+VEGF group. VEGF (50 μg/kg) was subcutaneously injected twice daily from d 1 to d 14. Mice in each group were killed at d 3, 7, or 14 after the operation, and the tubulointerstitial fibrosis was histopathologically evaluated. Human proximal tubular epithelial cells (HK-2) were used for in vitro study. The expression levels of α-SMA, E-cadherin, TGF-β1, CTGF, and BMP-7 in the kidney were determined using Western blot and RT-PCR.

Results:

In the UUO mice, the degree of interstitial fibrosis was dramatically increased in a time-dependent manner. At d 3, 7, and 14, both the mRNA and protein expression levels for α-SMA, TGF-β1, and CTGF were significantly upregulated, whereas those for E-cadherin and BMP-7 were significantly downregulated. At d 3 and 7, VEGF treatment significantly reduced interstitial fibrosis and the expression levels for α-SMA, TGF-β1, and CTGF, while significantly increased the expression of E-cadherin and BMP-7, as compared with the UUO mice. At d 14 after operation, no significant differences were observed in the expression of the examined markers between VEGF-treated mice and UUO mice, with the exception of CTGF. In HK-2 cells, VEGF blocked TGF-β1-induced α-SMA and vimentin expression and restored E-cadherin expression in a dose-dependent manner.

Conclusion:

VEGF may ameliorate renal tubulointerstitial fibrosis at the early stage in UUO mice. This effect may be related to inhibition of VEGF on renal tubular epithelial-mesenchymal transition (EMT).     

A novel, orally active LPA1 receptor antagonist inhibits lung fibrosis in the mouse bleomycin model

Background and purpose:

The aim of this study was to assess the potential of an antagonist selective for the lysophosphatidic acid receptor, LPA₁, in treating lung fibrosis We evaluated the in vitro and in vivo pharmacological properties of the high affinity, selective, oral LPA₁-antagonist (4′-{4-[(R)-1-(2-chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid (AM966).

Experimental approach:

The potency and selectivity of AM966 for LPA₁ receptors was determined in vitro by calcium flux and cell chemotaxis assays using recombinant and native cell cultures. The in vivo efficacy of AM966 to reduce tissue injury, vascular leakage, inflammation and fibrosis was assessed at several time points in the mouse bleomycin model.

Key results:

AM966 was a potent antagonist of LPA₁ receptors, with selectivity for this receptor over the other LPA receptors. In vitro, AM966 inhibited LPA-stimulated intracellular calcium release (IC₅₀ = 17 nM) from Chinese hamster ovary cells stably expressing human LPA₁ receptors and inhibited LPA-induced chemotaxis (IC₅₀ = 181 nM) of human IMR-90 lung fibroblasts expressing LPA₁ receptors. AM966 demonstrated a good pharmacokinetic profile following oral dosing in mice. In the mouse, AM966 reduced lung injury, vascular leakage, inflammation and fibrosis at multiple time points following intratracheal bleomycin instillation. AM966 also decreased lactate dehydrogenase activity and tissue inhibitor of metalloproteinase-1, transforming growth factor β1, hyaluronan and matrix metalloproteinase-7, in bronchoalveolar lavage fluid.

Conclusions and implications:

These findings demonstrate that AM966 is a potent, selective, orally bioavailable LPA₁ receptor antagonist that may be beneficial in treating lung injury and fibrosis, as well as other diseases that are characterized by pathological inflammation, oedema and fibrosis.     

Biphasic effect of arsenite on cell proliferation and apoptosis is associated with the activation of JNK and ERK1/2 in human embryo lung fibroblast cells

Biphasic dose-response relationship induced by environmental agents is often characterized with the effect of low-dose stimulation and high-dose inhibition. Some studies showed that arsenite may induce cell proliferation and apoptosis via biphasic dose-response relationship in human cells; however, mechanisms underlying this phenomenon are not well understood. In the present study, we aimed at investigating the relationship between biphasic effect of arsenite on cell proliferation and apoptosis and activation of JNK and ERK1/2 in human embryo lung fibroblast (HELF) cells. Our results demonstrated that cell proliferation may be stimulated at lower concentrations (0.1 and 0.5 microM) arsenite but inhibited at higher concentrations (5 and 10 microM). When cell apoptosis was used as the endpoint, the concentration-response curves were changed to U-shapes. During stimulation phospho-JNK levels were significantly increased at 3, 6, and 12 h after 0.1 or 0.5 microM arsenite exposure. Phospho-ERK1/2 levels were increased with different concentrations (0.1-10 microM) of arsenite at 6, 12, and 24 h. Blocking of JNK pathway with 20 microM SP600125 or ERK1/2 by 100 microM PD98059 significantly inhibited biphasic effect of arsenite in cells. Data in the present study suggest that activation of JNK and ERK1/2 may be involved in biphasic effect of arsenite when measuring cell proliferation and apoptosis in HELF cells. JNK activation seems to play a more critical role than ERK1/2 activation in the biphasic process.     

Potential role of c-Jun NH2-terminal kinase in allergic airway inflammation and remodelling: effects of SP600125

Asthma is a chronic inflammatory disease of the airways associated with structural changes such as increased airway smooth muscle mass, which may contribute to impairment of lung function. To determine whether c-Jun NH2-terminal kinase (JNK) of the mitogen-activated protein kinase signalling pathway participated in these changes, the effects of an inhibitor, SP600125 (anthra [1, 9-cd] pyrazole-6 (2H)-one), were examined in a murine model of chronic airway inflammation and remodelling. Mice sensitised to ovalbumin were exposed to ovalbumin aerosol and were treated with SP600125 [30 mg kg(-1) intraperitoneal (i.p.)] on days of exposure. SP600125 significantly reduced eosinophil and lymphocyte numbers in bronchoalveolar lavage fluid, suppressed eosinophilic inflammation within the bronchial submucosa, inhibited goblet cell hyperplasia, and increased airway smooth muscle cell number in allergen-exposed mice. SP600125 also inhibited allergen-induced increase in bronchial responsiveness. SP600125 inhibited JNK activity in the challenged lungs. Although SP 600125 may also have other effects, we conclude that c-Jun NH2-terminal kinase may play a role in allergen-induced inflammation and remodelling associated with bronchial hyperresponsiveness.     

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.     

Regulation of Bax by c-Jun NH2-terminal kinase and Bcl-xL in vinblastine-induced apoptosis

Microtubule inhibitors, such as vinblastine, are widely used in cancer chemotherapy. Vinblastine exerts its antitumor effect by inducing apoptosis. In KB-3 cells, we have shown previously that vinblastine activates c-Jun NH₂-terminal protein kinase (JNK) and causes Bax mitochondrial translocation and activation. In this study, we sought to test the hypothesis that JNK and Bcl-xL act as positive and negative regulators, respectively, of Bax translocation. The JNK inhibitor SP600125 inhibited vinblastine-induced JNK activation and in concert inhibited Bax mitochondrial translocation, Bax oligomerization, and Bax activation. Furthermore, the JNK inhibitor blocked vinblastine-induced apoptosis. The ability of vinblastine to induce Bax translocation and the inhibitory effect of SP600125 were confirmed in cells stably expressing GFP-Bax. However, if transiently overexpressed, Bax localized to the mitochondria, and this was associated with loss of viability and subsequent cell death. If Bcl-xL was co-expressed with Bax, the cells readily tolerated Bax overexpression. Indeed, physical interaction between Bcl-xL and Bax but not Bak was demonstrated by co-immunoprecipitation. These findings provide novel insight into the role of Bax and its regulation in vinblastine-induced apoptosis.     

不同ACEI类药物对糖尿病大鼠心肌间质纤维化的影响

目的通过研究卡托普利、贝那普利、福辛普利对糖尿病大鼠心肌转化生长因子β1（TGF—β1）及基质金属蛋白酶（MMPs）的影响,探讨这3种药物对糖尿病大鼠心肌的抗纤维化作用。方法50只SD大鼠分为10只正常组,40只糖尿病组,糖尿病组链脲佐菌素注射建模。建模成功后将成功的糖尿病鼠（37只,未成功2只,死亡1只）随机分糖尿病组（n=9）、卡托普利治疗组（n=9）、贝拉普利治疗组（n=9）、福辛普利治疗组（n=10）,免疫组化法测Ⅰ、Ⅲ型胶原的表达,TGF-β1、基质金属蛋白酶特异性抑制物（TIMP—1）和MMP-1蛋白的表达。RT—PCR法测TIMP-1和MMP-1 mRNA的表达。结果糖尿病组心脏指数心室指数显著大于正常组（P〈0．05）,Ⅰ、Ⅲ型胶原的表达也增加（P〈0．05）,存在着心肌间质纤维化,胶原Ⅰ、胶原Ⅲ、TGF—β1、TIMP-1蛋白及TIMP-1 mRNA的表达增高（P〈0．05）,MMP—1蛋白及mRNA的表达减少（P〈0．05）。经过卡托普利、福辛普利、贝那普利后,上述各异常指标均有所改善。结论3组药物通过对TGF-β1和MMPs的影响可明显改善糖尿病心肌间质纤维化。卡托普利的疗效比贝那普利与福辛普利稍差,贝那普利与福辛普利之间无差异。     

alpha1beta2delta, a silent GABAA receptor: recruitment by tracazolate and neurosteroids

BACKGROUND AND PURPOSE: This study investigated the alpha(1)beta(2)delta isoform of the GABA(A) receptor that is presumably expressed in the forebrain. The functional and pharmacological properties of this receptor combination are largely unknown. EXPERIMENTAL APPROACH: We expressed alpha(1)beta(2)delta GABA(A) receptors in Xenopus laevis oocytes. GABA-activated currents, in the presence and absence of modulators, were recorded using the two-electrode voltage clamp technique. KEY RESULTS: The alpha(1)beta(2)delta isoform of the GABA(A) receptor exhibited an extremely small GABA-mediated current. Tracazolate increased the current amplitude evoked by a half-maximal concentration (EC(50)) of GABA by 59-fold. The maximum current was increased 23-fold in the presence of a saturating GABA concentration. Concomitant with the increase in the maximum, was a 4-fold decrease in the EC(50). Finally, a mutation in the second transmembrane domain of the delta subunit that increases receptor efficacy (L286S), eliminated the increase in the maximum GABA-activated current. The endogenous neurosteroid, tetrahydrodeoxycorticosterone (THDOC), also decreased the EC(50) and increased the maximum current amplitude, although to a lesser degree than that of tracazolate. CONCLUSIONS AND IMPLICATIONS: Taken all together, these findings indicate that the small GABA-mediated currents in the absence of the modulator are due to a low efficacy for activation. In the absence of modulators, alpha(1)beta(2)delta GABA receptors would be effectively silent and therefore contribute little to inhibition in the CNS. In the presence of tracazolate or endogenous neurosteroids however, this particular receptor isoform could exert a profound inhibitory influence on neuronal activity.     

Nrf2 pathway activation contributes to anti-fibrosis effects of ginsenoside Rg1 in a rat model of alcohol- and CCl4-induced hepatic fibrosis

Aim:

To investigate the anti-fibrosis effects of ginsenoside Rg1 on alcohol- and CCl₄-induced hepatic fibrosis in rats and to explore the mechanisms of the effects.

Methods:

Rats were given 6% alcohol in water and injected with CCl₄ (2 mL/kg, sc) twice a week for 8 weeks. Rg1 (10, 20 and 40 mg/kg per day, po) was administered in the last 2 weeks. Hepatic fibrosis was determined by measuring serum biochemical parameters, HE staining, Masson''s trichromic staining, and hydroxyproline and α-SMA immunohistochemical staining of liver tissues. The activities of antioxidant enzymes, lipid peroxidation, and Nrf2 signaling pathway-related proteins (Nrf2, Ho-1 and Nqo1) in liver tissues were analyzed. Cultured hepatic stellate cells (HSCs) of rats were prepared for in vitro studies.

Results:

In the alcohol- and CCl₄-treated rats, Rg1 administration dose-dependently suppressed the marked increases of serum ALT, AST, LDH and ALP levels, inhibited liver inflammation and HSC activation and reduced liver fibrosis scores. Rg1 significantly increased the activities of antioxidant enzymes (SOD, GSH-Px and CAT) and reduced MDA levels in liver tissues. Furthermore, Rg1 significantly increased the expression and nuclear translocation of Nrf2 that regulated the expression of many antioxidant enzymes. Treatment of the cultured HSCs with Rg1 (1 μmol/L) induced Nrf2 translocation, and suppressed CCl₄-induced cell proliferation, reversed CCl_4- induced changes in MDA, GPX, PCIII and HA contents in the supernatant fluid and α-SMA expression in the cells. Knockdown of Nrf2 gene diminished these actions of Rg1 in CCl₄-treated HSCs in vitro.

Conclusion:

Rg1 exerts protective effects in a rat model of alcohol- and CCl₄-induced hepatic fibrosis via promoting the nuclear translocation of Nrf2 and expression of antioxidant enzymes.     

Genipin-induced apoptosis in hepatoma cells is mediated by reactive oxygen species/c-Jun NH2-terminal kinase-dependent activation of mitochondrial pathway

Genipin, the aglycone of geniposide, exhibits anti-inflammatory and anti-angiogenic activities. Here we demonstrate that genipin induces apoptotic cell death in FaO rat hepatoma cells and human hepatocarcinoma Hep3B cells, detected by morphological cellular changes, caspase activation and release of cytochrome c. During genipin-induced apoptosis, reactive oxygen species (ROS) level was elevated, and N-acetyl-l-cysteine (NAC) and glutathione (GSH) suppressed activation of caspase-3, -7 and -9. Stress-activated protein kinase/c-Jun NH2-terminal kinase 1/2(SAPK/JNK1/2) but neither MEK1/2 nor p38 MAPK was activated in genipin-treated hepatoma cells. SP600125, an SAPK/JNK1/2 inhibitor, markedly suppressed apoptotic cell death in the genipin-treated cells. The FaO cells stably transfected with a dominant-negative c-Jun, TAM67, was less susceptible to apoptotic cell death triggered by genipin. Diphenyleneiodonium (DPI), an inhibitor of NADPH oxidase, inhibited ROS generation, apoptotic cell death, caspase-3 activation and JNK activation. Consistently, the stable expression of Nox1-C, a C-terminal region of Nox1 unable to generate ROS, blocked the formation of TUNEL-positive apoptotic cells, and activation of caspase-3 and JNK in FaO cells treated with genipin. Our observations imply that genipin signaling to apoptosis of hepatoma cells is mediated via NADPH oxidase-dependent generation of ROS, which leads to downstream of JNK.     

Cadmium-induced apoptosis in the BJAB human B cell line: involvement of PKC/ERK1/2/JNK signaling pathways in HO-1 expression

Heme oxygenase-1 (HO-1, EC 1.14.99.3) is a key enzyme in the cellular response to tissue injury and oxidative stress. It oxidizes heme, a pro-oxidant and toxic species, to biliverdin, CO, and free iron. Cytoprotection during the heat shock response is a complex phenomenon involving multiple inducible mechanisms. Several important pathways involving serine/threonine kinases mediate the induction of HO-1 in response to external stimuli.     

Beneficial effects of pioglitazone against cardiovascular injury are enhanced by combination with aliskiren in a rat model of diabetic nephropathy

Objectives Aliskiren is the first in a new class of orally active direct renin inhibitors, approved for the treatment of hypertension. However, the efficacy of aliskiren in diabetic cardiovascular complications remains to be defined. This study aimed to test the hypothesis that aliskiren may enhance the beneficial effects of pioglitazone against cardiovascular injury associated with diabetic nephropathy. Methods Diabetic nephropathy was induced in rats by unilateral nephrectomy followed by streptozotocin injection. Diabetic nephropathic rats were orally given vehicle, pioglitazone, aliskiren, or combined pioglitazone and aliskiren for four weeks to compare their effects on cardiovascular injury, particularly myocardial fibrosis. Key findings Pioglitazone treatment significantly attenuated cardiac lipid peroxidation, oxidative injury and myocardial fibrosis in diabetic nephropathic rats. This was associated with up‐regulation of transforming growth factor‐β1 and matrix metalloproteinase‐2 genes, along with down‐regulation of tissue inhibitor of metalloproteinase‐2 gene in cardiac tissue. The combination of aliskiren with pioglitazone exerted greater beneficial effect than monotherapy with either drug, on all the aforementioned parameters. Conclusions Our findings suggested that aliskiren enhanced the protective effects of pioglitazone against myocardial fibrosis, in experimental diabetic nephropathy. Thus, the combination of aliskiren and pioglitazone may be a potential therapeutic strategy for cardiovascular injury associated with diabetic nephropathy.     

Mechanisms for prostaglandin E2 formation caused by proteinase-activated receptor-1 activation in rat gastric mucosal epithelial cells

Proteinase-activated receptor-1 (PAR1), a thrombin receptor, plays a protective role in gastric mucosa via prostanoid formation. Thus, we studied effects of PAR1 stimulation on prostaglandin E(2) (PGE(2)) formation in rat normal gastric mucosal epithelial RGM1 cells and analyzed the underlying signal transduction mechanisms. The PAR1-activating peptide (PAR1-AP) and thrombin increased PGE(2) release from RGM1 cells for 18h, an effect being suppressed by inhibitors of COX-1, COX-2, MEK, p38 MAP kinase (p38 MAPK), protein kinase C (PKC), Src and EGF receptor-tyrosine kinase (EGFR-TK), but not JNK and matrix metalloproteinase (MMP)/a disintegrin and metalloproteinases (ADAMs). PAR1-AP caused persistent (6h or more) and transient (5min) phosphorylation of ERK and p38 MAPK, respectively, followed by delayed reinforcement at 18h. PAR1-AP up-regulated COX-2 in a manner dependent on MEK and EGFR-TK, but not p38 MAPK. The PAR1-mediated persistent ERK phosphorylation was reduced by inhibitors of Src and EGFR-TK. PAR1-AP actually phosphorylated EGF receptors and up-regulated mRNA for heparin-binding-EGF (HB-EGF), the latter effect being blocked by inhibitors of Src, EGFR-TK and MEK. Heparin, an inhibitor for HB-EGF, suppressed PAR1-mediated PGE(2) formation and persistent ERK phosphorylation. These results suggest that PAR1 up-regulates COX-2 via persistent activation of MEK/ERK that is dependent on EGFR-TK activation following induction of HB-EGF, leading to PGE(2) formation. In addition, our data also indicate involvement of COX-1, PKC and p38 MAPK in PAR1-triggered PGE(2) formation. PAR1, thus stimulates complex multiple signaling pathways responsible for PGE(2) formation in RGM1 cells.     

How biologics targeting the IL-1 system are being considered for the treatment of type 2 diabetes

Metabolic diseases are associated with activation of the innate immune system in various tissues and characterized by elevated inflammatory factors and the presence of immune cells. Type 2 diabetes develops when islet beta cells are deficient in producing sufficient insulin to overcome peripheral insulin resistance. Intra-islet IL-1β activity diminishes beta cell function and survival and governs islet inflammation. Targeting the IL-1 system with the IL-1 receptor antagonist IL1Ra improved insulin secretion, glycaemia and reduced systemic inflammation in a proof of concept study with patients with type 2 diabetes. Currently, long lasting and specific IL-1β blocking antibodies are being evaluated in clinical trials and this may lead to a novel cytokine-based treatment for type 2 diabetes.