Cistanches Herba aqueous extract affecting serum BGP and TRAP and bone marrow Smad1 mRNA, Smad5 mRNA, TGF-β1 mRNA and TIEG1 mRNA expression levels in osteoporosis disease

We studied molecular mechanism of Cistanches Herba aqueous extract (CHAE) in ovariectomized (OVX) rats, as an experimental model of postmenopausal osteoporosis. Female rats were either sham-operated or bilaterally OVX; and at 60 days postoperatively. The OVX group (n = 8) received an ovariectomy and treatment with normal saline for 90 days commencing from 20th post ovariectomy day. The ovariectomized +CHAE (OVX + CHAE) group (n = 8) received an ovariectomy and were treated with Cistanches Herba aqueous extract of 100 mg/kg body weight daily for 90 days commencing from 22nd post ovariectomy day. The ovariectomy +CHAE (OVX + CHAE) group (n = 8) received an ovariectomy, and were treated with the of 200 mg/kg body weight daily for 90 days commencing from 20th post ovariectomy day. Serum BGP and TRAP, E2, FSH and LH level, bone marrow Smad1, Smad5, TGF-β1 and TIEG1 mRNA expression levels were examined. Results showed that serum BGP and TRAP, FSH and LH levels were significantly increased, whereas E2, Smad1, Smad5, TGF-β1 and TIEG1 mRNA and proteins expression levels were significantly decreased in OVX rats compared to sham rats. 90 days of CHAE treatment could significantly decrease serum BGP and TRAP, FSH and LH levels, and increase E2, Smad1, Smad5, TGF-β1 and TIEG1 mRNA and proteins expression levels in OVX rats. It can be concluded that CHAE play its protective effect against OVX-induced bone degeneration partly by regulating some bone metabolism related genes, e.g. Smad1, Smad5, TGF-β1 and TIEG1.     

Characterization of Smad3 knockout mouse derived skin cells

TGF-β plays an important role in skin wound healing process, in which Smad3 acts as a signaling molecule. Smad3 knockout mice exhibit enhanced wound healing and less inflammatory process, but the intrinsic properties of the mouse derived skin cells are generally unexplored. The purpose of this study is to characterize the biological behavior of skin cells derived from Smad3 knockout mice and thus to define the mechanism of this particular wound healing process. Keratinocytes and dermal fibroblasts were harvested from the skin of Smad3 knockout (Smad3 KO) and wild-type (WT) mice and in vitro cultured for one and two passages for various experiments. The results showed that KO mouse serum contained significantly higher levels of TGF-β1 and lower level of IL-6 and IL-10 than WT mouse serum (p < 0.05), which were also supported by the same findings of more TGF-β1 and less IL-6 and IL-10 in the supernatant of cultured KO dermal fibroblasts than those of WT cells (p < 0.05). At gene levels, IL-6, IL-10, and TGF-β1 were significantly less expressed in KO fibroblasts than in WT fibroblasts (p < 0.05). In addition, KO dermal fibroblasts also exhibited stronger migration and proliferation potentials than WT fibroblasts (p < 0.05). Moreover, both KO fibroblasts and keratinocytes showed higher colony-forming efficiency than WT counterparts with significant difference (p < 0.05). These findings indicate that both systemic factors and intrinsic properties of skin cells contribute to enhanced wound healing and less inflammatory reaction observed in Smad3 knock-out mice.     

Is cytochrome P450 3A4 regulated by menstrual cycle hormones in control endometrium and endometriosis?

The aim of this study is to investigate the effect of evodiamine on fibroblast activation in cardiac fibroblasts and endothelial to mesenchymal transition (EndMT) in human umbilical vein endothelial cells (HUVECs). Neonatal rat cardiac fibroblasts were stimulated with transforming growth factor beta 1 (TGF-β1) to induce fibroblast activation. After co-cultured with evodiamine (5, 10 μM), the proliferation and pro-fibrotic proteins expression of cardiac fibroblasts were evaluated. HUVECs were also stimulated with TGF-β1 to induce EndMT and treated with evodiamine (5, 10 μM) at the same time. The EndMT response in the HUVECs was evaluated as well as the capacity of the transitioned endothelial cells migrating to surrounding tissue. As a result, Evodiamine-blunted TGF-β1 induced activation of cardiac fibroblast into myofibroblast as assessed by the decreased expressions of α-SMA. Furthermore, evodiamine reduced the increased protein expression of fibrosis markers in neonatal and adult rat cardiac fibroblasts induced by TGF-β1. HUVECs stimulated with TGF-β1 exhibited lower expression levels of CD31, CD34, and higher levels of α-SMA, vimentin than the control cells. This phenotype was eliminated in the HUVECs treated with both 5 and 10 μM evodiamine. Evodiamine significantly reduced the increase in migration ability that occurred in response to TGF-β1 in HUVECs. In addition, the activation of Smad2, Smad3, ERK1/2, and Akt, and the nuclear translocation of Smad4 in both cardiac fibroblasts and HUVEC were blocked by evodiamine treatment. Thus, evodiamine could prevent cardiac fibroblasts from activation into myofibroblast and protect HUVEC against EndMT. These effects may be mediated by inhibition of the TGFβ pathway in both cardiac fibroblasts and HUVECs.     

Periodontal ligament-associated protein-1 knockout mice regulate the differentiation of osteoclasts and osteoblasts through TGF-β1/Smad signaling pathway

It has been known that periodontal ligament-associated protein-1 (PLAP-1/Asporin) not only inhibits cartilage formation in osteoarthritis, but it also influences the healing of skull defect. However, the effect and mechanism of PLAP-1/Asporin on the mutual regulation of osteoclasts and osteoblasts in periodontitis are not clear. In this study, we utilized a PLAP-1/Asporin gene knockout (KO) mouse model to research this unknown issue. We cultured mouse bone marrow mesenchymal stem cells with Porphyromonas gingivalis lipopolysaccharide (P.g. LPS) for osteogenic induction in vitro. The molecular mechanism of PLAP-1/Asporin in the regulation of osteoblasts was detected by immunoprecipitation, immunofluorescence, and inhibitors of signaling pathways. The results showed that the KO of PLAP-1/Asporin promoted osteogenic differentiation through transforming growth factor beta 1 (TGF-β1)/Smad3 in inflammatory environments. We further found the KO of PLAP-1/Asporin inhibited osteoclast differentiation and promoted osteogenic differentiation through the TGF-β1/Smad signaling pathway in an inflammatory coculture system. The experimental periodontitis model was established by silk ligation and the alveolar bone formation in PLAP-1/Asporin KO mice was promoted through TGF-β1/Smad3 signaling pathway. The subcutaneous osteogenesis model in nude mice also confirmed that the KO of PLAP-1/Asporin promoted bone formation by the histochemical staining. In conclusion, PLAP-1/Asporin regulated the differentiation of osteoclasts and osteoblasts through TGF-β1/Smad signaling pathway. The results of this study lay a theoretical foundation for the further study of the pathological mechanism underlying alveolar bone resorption, and the prevention and treatment of periodontitis.     

NLRP1 promotes TGF-β1-induced myofibroblast differentiation in neonatal rat cardiac fibroblasts

Nuclear localization leucine-rich-repeat protein 1 (NLRP1) is a member of Nod-like receptors (NLRs) family. Recent studies have reported that NLRP1 is involved in various diseases, especially in cardiovascular diseases. However, the effect of NLRP1 on cardiac fibrosis remains unclear. In this study, NLRP1 overexpression and NLRP1 silencing constructs were transfected into neonatal rat cardiac fibroblasts induced by TGF-β1 for 48 h to investigate the effect of NLRP1 in cardiac fibrosis and its molecular mechanisms. Cardiac fibroblasts were transfected with NLRP1 and then cultured in the presence and absence of TGF-β1and Smad3 inhibitor (SIS3). Our data indicated that NLRP1 not only promoted fibroblast activation and myofibroblast differentiation, but also upregulated the mRNA and protein levels of α-SMA in the TGF-β1-treated neonatal rat cardiac fibroblasts. Overexpressing NLRP1 in TGF-β1-induced cardiac fibroblasts upregulated the mRNA and protein levels of Collagen I, Collagen III, and connective tissue growth factor. Moreover, NLRP1 upregulated the protein levels of Smad2, Smad3, and Smad4 in nuclei of fibroblasts, and attenuated levels of phosphorylated Smad2 and Smad3 in the cytoplasm of fibroblasts induced by TGF-β1. In addition, the increase in fibrotic genes and Smad proteins was significantly reduced in the presence of SIS3. Our findings illustrated that NLRP1 promoted myofibroblast differentiation and excessive ECM production in TGF-β1-induced neonatal cardiac fibroblasts through directly targeting TGF-β1/Smad signaling pathways.     

紫草油治疗肛瘘术后的临床研究

目的观察紫草油对肛瘘术后临床疗效改善情况以及血清中TGF-β1、Smad3的含量变化。方法将我院收治的肛瘘术后患者90例采用简单随机法分为紫草油组、凡士林组和肤痔清组各30例,术后第1天至创面愈合,每天分别给予紫草油、凡士林、肤痔清软膏外用,并于术后第1、7、14、21天观察创面疼痛、水肿、出血情况,以及酶联免疫吸附法(ELISA法)检测患者血清中TGF-β1和Smad3含量。结果 3组患者治疗后均能缓解术后水肿、疼痛、出血症状;紫草油组在治疗后第21天与肤痔清组、凡士林组比较,水肿、疼痛、出血的积分差异显著(P0.05)。治疗后第7、14、21天紫草油组与肤痔清组、凡士林组的TGF-β1含量比较,差异具有统计学意义(P0.05);治疗后组内血清Smad3含量均较治疗前明显降低,差异有统计学意义(P0.05),但治疗后第21天紫草油组与肤痔清组、凡士林组的Smad3含量比较,差异无统计学意义(P0.05)。结论紫草油能明显缓解肛瘘术后创面水肿、疼痛、出血情况,且疗效优于肤痔清及凡士林,能有效提高TGF-β1含量及降低血清Smad3含量,促进创面愈合。     

The ERK/CREB pathway is involved in the c-Ski expression induced by low TGF-β1 concentrations during primary fibroblast proliferation

Increasing evidence has suggested that bidirectional regulation of cell proliferation is one important effect of TGF-β1 in wound healing. Increased c-Ski expression plays a role in promoting fibroblast proliferation at low TGF-β1 concentrations, but the mechanism by which low TGF-β1 concentrations regulate c-Ski levels remains unclear. In this study, the proliferation of rat primary fibroblasts was assessed with an ELISA BrdU kit. The mRNA and protein expression and phosphorylation levels of corresponding factors were measured by RT-qPCR, immunohistochemistry or Western blotting. We first found that low TGF-β1 concentrations not only promoted c-ski mRNA and protein expression in rat primary fibroblasts but also increased the phosphorylation levels of Extracellular Signal-Regulated Kinases (ERK) and cAMP response element binding (CREB) protein. An ERK kinase (mitogen-activated protein kinase kinase, MEK) inhibitor significantly inhibited ERK1/2 phosphorylation levels, markedly reducing c-Ski expression and CREB phosphorylation levels and abrogating the growth-promoting effect of low TGF-β1 concentrations. At the same time, Smad2/3 phosphorylation levels were not significantly changed. Taken together, these results suggest that the increased cell proliferation induced by low TGF-β1 concentrations mediates c-Ski expression potentially through the ERK/CREB pathway rather than through the classic TGF-β1/Smad pathway.     

Impaired TGF-β signaling and a defect in resolution of inflammation contribute to delayed wound healing in a female rat model of type 2 diabetes

Wound healing (WH) impairment is a well-documented phenomenon in clinical and experimental diabetes. Sex hormones, in addition to a number of signaling pathways including transforming growth factor-β1 (TGF-β1)/Smads and TNF-α/NF-κB in macrophages and fibroblasts, appear to play a cardinal role in determining the rate and nature of WH. We hypothesized that a defect in resolution of inflammation and an enhancement in TNF-α/NF-κB activity induced by estrogen deficiency contribute to the impairment of TGF-β signaling and delayed WH in diabetes models. Goto-Kakizaki (GK) rats and full thickness excisional wounds were used as models for type 2 diabetes (T2D) and WH, respectively. Parameters related to the various stages of WH were assessed using histomorphometry, western blotting, real-time PCR, immunofluorescence microscopy and ELISA-based assays. Retarded re-epithelialization, suppressed angiogenesis, delayed wound closure, reduced estrogen level and heightened states of oxidative stress were characteristic features of T2D wounds. These abnormalities were associated with a defect in resolution of inflammation, shifts in macrophage phenotypes, increased β3-integrin expression, impaired wound TGF-β1 signaling (↓p-Smad2/↑Smad7) and enhanced TNF-α/NFκB activity. Human/rat dermal fibroblasts of T2D, compared to corresponding control values, displayed resistance to TGF-β-mediated responses including cell migration, myofibroblast formation and p-Smad2 generation. A pegylated form of soluble TNF receptor-1 (PEG-sTNF-RI) or estrogen replacement therapy significantly improved re-epithelialization and wound contraction, enhanced TGFβ/Smad signaling, and polarized the differentiation of macrophages toward an M2 or "alternatively" activated phenotype, while limiting secondary inflammatory-mediated injury. Our data suggest that reduced estrogen levels and enhanced TNF-α/NF-κB activity delayed WH in T2D by attenuating TGFβ/Smad signaling and impairing the resolution of inflammation; most of these defects were ameliorated with estrogen and/or PEG-sTNF-RI therapy.     

Anti-Inflammatory Effects of Ginsenoside-Rh2 Inhibits LPS-Induced Activation of Microglia and Overproduction of Inflammatory Mediators Via Modulation of TGF-β1/Smad Pathway

Microglia activation plays an important role in neuroinflammation and contributes to several neurological disorders. Hence, inhibition of both microglia activation and pro-inflammatory cytokines may lead to the effective treatment of neurodegenerative diseases. In this study, we found that GRh2 inhibited the inflammatory response to lipopolysaccharide (LPS) and prevented the LPS-induced neurotoxicity in microglia cells. GRh2 significantly decreased the generation of nitric oxide production, and tumor necrosis factor-α, interleukin (IL)-6, IL-1β, cyclooxygenase-2 and inducible nitric oxide synthase in LPS-induced activated microglia cells. Furthermore, GRh2 (20 and 50 μM) significantly increased TGF-β1 expression and reduced the expression of Smad. These results suggest that GRh2 effectively inhibits microglia activation and production of pro-inflammatory cytokines via modulating the TGF-β1/Smad pathway.     

Thrombospondin-4 critically controls transforming growth factor β1 induced hypertrophic scar formation

Transforming growth factor β (TGF-β) is a growth factor presenting important functions during tissue remodeling and hypertrophic scar (HS) formation. However, the underlying molecular mechanisms are largely unknown. In this study, we identified thrombospondin-4 (TSP-4) as a TGF-β1 target that essentially mediates TGF-β1-induced scar formation both in vitro and in vivo. The expression of TSP-4 was compared on both mRNA and protein levels between hypertrophic scar fibroblasts (HSFs) and normal skin fibroblast (NFs) in response to TGF-β1 treatment. Two signaling molecules, Smad3 and p38, were assessed for their importance in regulating TGF-β1-mediated TSP-4 expression. The significance of TSP-4 in controlling TGF-β1-induced proliferation, invasion, migration, and fibrosis in HSFs was analyzed by knocking down endogenous TSP-4 using small hairpin RNA (shRNA) (TSP-4 shRNA). Finally, a skin HS model was established in rats and the scar formation was compared between rats treated with vehicle (saline), TGF-β1, and TGF-β1 + TSP-4 shRNA. The TSP-4 level was significantly higher in HSFs than in NFs and TGF-β1 more potently boosted TSP-4 expression in the former than in the latter. Both Smad3 and p38 essentially mediated TGF-β1-induced TSP-4 expression. TSP-4 shRNA significantly suppressed TGF-β1-stimulated proliferation, invasion, migration, or fibrosis of HSFs in vitro and drastically improved wound healing in vivo. TGF-β1, by activating both Smad3 and p38, induces TSP-4, which in turn not only presents a positive feedback regulation on the activation of Smad3 and p38, but also essentially mediates TGF-β1-induced HS formation. Targeting TSP-4 thus may benefit HS treatment.     

胎盘间充质干细胞治疗小鼠烫伤愈合的相关研究

目的:观察胎盘间充质干细胞对TGF-β1/Smad信号通路的调控作用,探讨胎盘间充质干细胞对烫伤愈合及瘢痕形成的影响。方法:构建小鼠烫伤模型,注射人胎盘间充质干细胞(hPMSCs),荧光显微镜观察小鼠创伤皮肤组织中hPMSCs细胞的存活情况;HE和Masson染色观察小鼠创伤皮肤的变化;Western blot检测观察创伤皮肤TGF-β1、p-Smad3、Smad7、α-SMA、collagen I、Collagen III蛋白表达变化。结果:注射hPMSCs细胞后,小鼠创伤面积逐渐减小,创伤愈合率逐渐增加;hPMSCs细胞分布在小鼠创伤皮肤组织中,存活状况较好。进一步研究发现烫伤模型组皮肤表层细胞受损脱落,真皮层组织疏松,毛囊、皮脂腺等附属器坏死,可见明显的毛细血管扩张,并伴有炎性细胞渗出,同时可见大量的成纤维细胞增生和胶原纤维形成;注射hPMSCs细胞治疗后,病理改变、纤维增生和胶原形成明显减轻;此外,烫伤模型组创伤皮肤组织中TGF-β1、p-Smad3表达明显上调,Smad7蛋白表达明显下调,α-SMA、collagen I、Collagen III表达明显上调。经hPMSCs细胞治疗后,TGF-β1、p-Smad3蛋白表达明显下调,Smad7蛋白表达明显上调,α-SMA、collagen I、Collagen III蛋白表达明显下调。结论:胎盘间充质干细胞可能通过抑制TGF-β1/Smad信号通路,发挥促进烫伤愈合且抑制瘢痕形成的作用。     

糖皮质激素抑制结肠癌细胞增殖和侵袭的作用机制

本研究目的是为了证实地塞米松对结肠癌LoVo细胞增殖的抑制作用,并阐明其中的分子机制。LoVo细胞经不同浓度梯度地塞米松干预,再加入TGF-β1受体抑制剂SB431542阻断TGF-β1信号传导途径,通过MTS分析各组细胞增殖情况,借助Hoechst 33342和Annexin V/PI染色法检测细胞凋亡率;结合Western blotting对TGF-β1、Smad2和caspase-3蛋白表达情况的检测结果,分析地塞米松诱导结肠癌LoVo细胞凋亡的作用机理。LoVo细胞在1.0 mmol/L和10.0 mmol/L地塞米松干预48 h后,细胞增殖率与对照组相比分别降低32%(p<0.01)和47%(p<0.001),2组细胞凋亡率分别为28%和36%(p<0.001)。Western blotting结果显示,与对照组相比,地塞米松以浓度依赖性方式显著上调LoVo细胞TGF-β1、Smad2和Cleavedcaspase-3蛋白水平(p<0.01),而TGF-β1受体抑制剂SB431542明显下调TGF-β1、Smad2和Cleaved-capase-3蛋白表达(p<0.05)。流式细胞术检测结果表明,SB431542+地塞米松干预组与地塞米松处理组LoVo细胞凋亡率分别为8%和23%(p<0.001)。地塞米松可显著诱导LoVo细胞凋亡,而SB431542能够挽救这一过程,这表明,地塞米松通过TGF-β1/Smad2通路诱导LoVo细胞凋亡。     

血管平滑肌细胞中ERK通路与TGF-β_1/Smad通路的相互作用

目的:探讨血管平滑肌细胞(VSMC)中TGF-β/Smad与ERK信号转导通路是否存在相互调节关系。方法:原代培养的大鼠胸主动脉平滑肌细胞,分四组:①对照组,②TGF-β1组,③ERK阻断剂(PD98059)组和④TGF-β1+ERK阻断剂(PD98059)组。分别用Western blot法检测VSMC内Smad2/3、ERK1/2蛋白表达及磷酸化Smad2/3、磷酸化ERK1/2蛋白含量,RT-PCR方法测VSMC中Smad2、Smad3mRNA的表达。结果:①与对照组相比,TGF-β1组P-Smad2/3、P-ERK1/2蛋白含量增多(P0.05),ERK阻断剂组P-Smad2/3、P-ERK1/2蛋白含量减少(P0.05),TGF-β1+ERK阻断剂组P-Smad2/3、P-ERK1/2蛋白含量无差异;与TGF-β1组相比,TGF-β1+ERK阻断剂组P-Smad2/3、P-ERK1/2蛋白含量减少(P0.05)。各组间Smad2/3、ERK1/2蛋白表达无差异。②各组的Smad2、Smad3mRNA表达无差异。结论:TGF-β1诱导的Smad2/3蛋白磷酸化依赖ERK通路激活,但ERK通路对Smad2/3蛋白和mRNA表达水平无影响。     

Bushenhuoxue formula accelerates fracture healing via upregulation of TGF-β/Smad2 signaling in mesenchymal progenitor cells

BackgroundAlthough Bushenhuoxue formula (BSHXF) is successfully used as a non-traumatic therapy in treating bone fracture in China, the molecular mechanism underlying its effects remains poorly understood.PurposeThe present study aims to explore the therapeutic effects of BSHXF on fracture healing in mice and the underlying mechanism.MethodsWe performed unilateral open transverse tibial fracture procedure in C57BL/6 mice which were treated with or without BSHXF. Fracture callus tissues were collected and analyzed by X-ray, micro-CT, biomechanical testing, histopathology and quantitative gene expression analysis. Tibial fracture procedure was also performed in Cre-negative and Gli1-CreER; Tgfbr2flox/flox conditional knockout (KO) mice (Tgfbr2^Gli1ER) to determine if BSHXF enhances fracture healing in a TGF-β-dependent manner. In addition, scratch-wound assay and cell counting kit-8 (CCK-8) assay were used to evaluate the effect of BSHXF on cell migration and cell proliferation in C3H10T1/2 mesenchymal stem cells, respectively.ResultsBSHXF promoted endochondral ossification and enhanced bone strength in wild-type (WT) or Cre- control mice. In contrast, BSHXF failed to promote bone fracture healing in Tgfbr2^Gli1ER conditional KO mice. In the mice receiving BSHXF treatment, TGF-β/Smad2 signaling was significantly activated. Moreover, BSHXF enhanced cell migration and cell proliferation in C3H10T1/2 cells, which was strongly attenuated by the small molecule inhibitor SB525334 against TGF-β type I receptor.ConclusionThese data demonstrated that BSHXF promotes fracture healing by activating TGF-β/Smad2 signaling. BSHXF may be used as a type of alternative medicine for the treatment of bone fracture healing.     

Lefty A attenuates the TGF-β1-induced epithelial to mesenchymal transition of human renal proximal epithelial tubular cells

The epithelial to mesenchymal transition (EMT) is a crucial event for renal fibrosis that can be elicited by TGF-β1/Smads signaling and its downstream mediator connective tissue growth factor (CTGF). As a distinct member of the TGF-β superfamily, Lefty A has been shown to be significantly downregulated in the kidneys of patients with severe ureteral obstruction, suggesting its role in renal fibrosis induced by obstructive nephropathy. In order to determine whether Lefty A prevents TGF-β1-induced EMT, human proximal tubule epithelial cells (HK-2) were stably transfected with Lefty A or control vectors and stimulated with 10 ng/ml TGF-β1 for 48 h. The results show that stimulation with TGF-β1 led to EMT including cell morphology changes, Smad2/3 signaling pathway activation, increased α-SMA, collagen type I, and CTGF expression, and decreased E-cadherin expression in mock-transfected HK-2 cells. Overexpression of Lefty A efficiently blocked p-Smad2/3 activation and attenuated all these EMT changes induced by TGF-β1. This finding suggests that Lefty A may serve as a potential new therapeutic target to inhibit or even reverse EMT during the process of renal fibrosis.     

多配体蛋白多糖-1在慢性阻塞性肺疾病大鼠肺上皮间质转化中的作用及机制*

目的:探讨多配体蛋白多糖-1(syndecan-1)在慢性阻塞性肺疾病(COPD)大鼠肺上皮间质转化(EMT)中的作用及机制。方法:选择清洁级SD雄性大鼠30只,随机分为假手术组(暴露气管后注射生理盐水,n=10),COPD组(注射1 mg/ml脂多糖后进行烟熏,造模完成后转染100 μl空载病毒,n=10),syndecan-1过表达组(注射1 mg/ml脂多糖后进行烟熏。造模完成后转染100 μl携带大鼠syndecan-1基因的重组腺病毒载体Ad-CMV-GFP-SDC1,n=10),每天1次,连续处理2周。处理结束后检测各组大鼠肺功能并取肺组织;采用HE染色法观察肺组织损伤情况;采用免疫组化检测各组大鼠肺组织syndecan-1、波形蛋白(vimentin)、E-钙黏蛋白(E-cadherin)的蛋白表达;采用Western blot检测转化生长因子-β1(TGF-β1)、Smad2/3和p-Smad2/3蛋白表达水平;采用qRT-PCR法检测各组大鼠肺组织中vimentin、E-cadherin、TGF-β1和Smad2/3 mRNA表达水平。结果:与假手术组相比,COPD组大鼠气道黏膜脱落,管腔狭窄,气道壁较多炎性细胞浸润,肺气肿严重,每分钟呼气量(V_E)、最大呼气流量(PEF)和0.3s用力呼气容积(FEV_0.3)和肺组织E-cadherin mRNA表达水平均显著降低(P＜0.05),而vimentin、TGF-β1、Smad2/3 mRNA表达水平及TGF-β1、Smad2/3和p-Smad2/3的蛋白表达水平均显著升高(P＜0.05)。与COPD组相比,syndecan-1过表达组气道黏膜脱落及气道壁炎性细胞浸润数减少,管腔狭窄、肺气肿情况均有所改善,V_E、PEF、FEV_0.3和肺组织E-cadherin mRNA表达水平显著升高(P＜0.05),而vimentin、TGF-β1、Smad2/3 mRNA表达水平及TGF-β1、Smad2/3和p-Smad2/3的蛋白表达水平均显著降低(P＜0.05)。结论:COPD大鼠体内TGF-β/Smad信号通路活化且存在肺EMT;过表达syndecan-1可抑制TGF-β/Smad信号通路,减轻EMT,改善COPD大鼠肺组织损伤。     

Alcohol-induced increase in BMP levels promotes fatty liver disease in male prediabetic stage Otsuka Long-Evans Tokushima Fatty rats

Alcohol consumption exacerbates liver abnormalities in animal models, but whether it increases the severity of liver disease in early diabetic or prediabetic rats is unclear. To investigate the molecular mechanisms underlying alcohol-induced liver steatosis or hepatitis, we used a prediabetic animal model. Otsuka Long-Evans Tokushima Fatty (OLETF) and Long-Evans Tokushima Fatty (LETO) rats were pair-fed with an ethanol-containing liquid diet for 6 weeks. Compared with controls, OLETF and LETO rats displayed more pronounced liver steatosis and higher plasma levels of serum glutamic oxaloacetic transaminase (SGOT) and serum glutamate pyruvate transaminase (SPGT), indicating liver injury. Ethanol-fed LETO (Pd-L-E) rats showed mild liver steatosis and no inflammation compared with ethanol-fed OLETF (Pd-O-E) rats. Although precursor and active SREBP-1 levels in the liver of ethanol-fed OLETF rats significantly increased compared with control diet-fed OLETF rats (Pd-O-C), those of Pd-L-E rats did not. Bone morphogenetic protein (BMP) and TGF-β1 balance in Pd-O-E rats was significantly altered because BMP signaling was upregulated by inducing BMP2, BMP4, BMP7, BMP9, Smad1, and Smad4, whereas TGF-β1, Smad3, and Erk were downregulated. Activation of TGF-β/Smad signaling inhibited BMP2 and BMP9 expression and increased epithelial-mesenchymal transition (EMT) marker levels (Hepcidin, Snail, and Twist) in the liver of LETO rats. Livers of ethanol-fed OLETF rats showed increased levels of vimentin, FSP-1, α-SMA, MMP1, MMP13, and collagen III compared with rats of other groups, whereas EMT marker levels did not change. Thus, BMP exerted anti- and/or pro-fibrotic effects in ethanol-fed rats. Therefore, BMP and TGF-β, two key members of the TGF-β superfamily, play important but diverse roles in liver steatosis in young LETO and OLETF rats.     

异甘草酸镁对纤维化大鼠肝脏TGF-β 1及Smad蛋白表达的影响

目的：观察异甘草酸镁对四氯化碳诱导的肝纤维化大鼠肝脏组织TGF-β1及Smad蛋白表达的影响,以期揭示其抗纤维化的机制。方法：利用腹腔注射四氯化碳（CCl4）建立大鼠肝纤维化模型,然后应用不同剂量的异甘草酸镁和INF-γ处理,于实验第16周末检测大鼠血清透明质酸（HA）、层粘连蛋白（LN）、III型前胶原（PC-III）、IV型胶原（C-IV）的水平,采用RT-PCR法检测TGF-β1,Smad3,Smad7mRNA的表达。结果：与模型组比较,异甘草酸镁各剂量组血清HA,LN,PC-III,C-IV水平显著下降（P〈0.05）,肝脏TGF-β1、smad3的表达明显降低（P〈0.05）,smad7则有所上升。结论：异甘草酸镁可以改善肝纤维化大鼠肝组织纤维化程度,其作用机理与抑制TGF-β1、Smad3mRNA的表达,上调Smad7mRNA的表达有密切关系。     

调节TGF-β1/Smad信号通路对内质网应激状态下肝癌HepG2细胞凋亡的影响

目的: 探讨TGF-β1/Smad信号通路对内质网应激(ERS)状态下肝癌HepG2细胞凋亡的影响机制。方法: 首先建立内质网应激模型:以3 μmol/L的衣霉素(TM)处理人肝癌HepG2细胞株24 h,诱导细胞发生ERS。实验分为6组,每组3个复孔,实验重复3次,6组分别为:Untreated组(未处理组)、TM组(3 μmol/L TM处理组)、TM+NC组(3 μmol/L TM+si-TGF-β1阴性对照组)、TM+si-TGF-β1组(3 μmol/L TM+si-TGF-β1组)、TM+pEX-3组(3 μmol/L TM+质粒对照组)及TM+TGF-β1 pEX-3组(3 μmol/L TM+TGF-β1过表达质粒组),利用脂质体的方法将TGF-β1小干扰RNA(si-TGF-β1)及TGF-β1过表达质粒(TGF-β1 pEX-3)转染入HepG2细胞,转染24 h后,利用RT-qPCR和Western blot检测各组HepG2细胞TGF-β1/Smad信号通路相关因子TGF-β1、p-Smad2表达的情况;CCK-8和流式细胞术分别检测各组HepG2细胞增殖抑制率和凋亡率变化情况。结果: 与Untreated组相比,TM组细胞的TGF-β1及p-Smad2的表达明显降低(P＜0.05);与TM组相比,TM+si-TGF-β1组细胞的TGF-β1及p-Smad2的表达和细胞的增殖抑制率、凋亡率显著降低(P＜0.01),而TM+TGF-β1 pEX-3组细胞的TGF-β1及p-Smad2的表达和细胞增殖抑制率、凋亡率显著升高(P＜0.01)。结论: TGF-β1/Smad信号通路在肝癌HepG2细胞发生ERS后受到抑制,当该通路被激活后,ERS状态下肝癌HepG2细胞的凋亡率显著升高。