Protective effects of total flavonoids from Qu Zhi Qiao (fruit of Citrus paradisi cv. Changshanhuyou) on OVA-induced allergic airway inflammation and remodeling through MAPKs and Smad2/3 signaling pathway.

Allergic asthma is one of the inflammatory diseases, which has become a major public health problem. Qu zhi qiao (QZQ), a dry and immature fruit of Citrus paradisi cv. Changshanhuyou, has various flavonoids with pharmacological properties. However, there is a knowledge gap on the pharmacological properties of QZQ on allergic asthma. Therefore, here, we explored the efficacy and mechanism of total flavonoids from QZQ (TFCH) on allergic asthma. We extracted and purified TFCH and conducted animal experiments using an Ovalbumin (OVA)-induced mice model. Bronchoalveolar lavage fluid and Swiss-Giemsa staining were used to count different inflammatory cells in allergic asthma mice. We conducted histopathology and immunohistochemistry to evaluate the changes in the lungs of allergic asthma mice. Moreover, we used ELISA assays to analyze chemokines and inflammatory cytokines. Furthermore, western blot analyses were conducted to elucidate the mechanism of TFCH on allergic asthma. We established that TFCH has anti-inflammatory effects and inhibits airway remodeling, providing a potential therapeutic strategy for allergic asthma.

Xiaoyaosan decoction alleviated rat liver fibrosis via the TGFß/Smad and Akt/FoxO3 signaling pathways based on network pharmacology analysis.

ETHNOPHARMACOLOGICAL RELEVANCE: Liver fibrosis is an outcome of many chronic liver diseases and often results in cirrhosis, liver failure, and even hepatocarcinoma. Xiaoyaosan decoction (XYS) as a classical Traditional Chinese Medicine (TCM) formula is used to liver fibrosis in clinical practice while its mechanism is unclear. AIM OF THE STUDY: The aim of this study was to investigate the anti-fibrosis effect of XYS and to explore the molecular mechanisms by combining network pharmacology and transcriptomic technologies. MATERIALS AND METHODS: The carbon tetrachloride (CCl4)-induced liver fibrosis rat were treated with three doses of XYS. The liver fibrosis and function were evaluated by histopathological examination and serum biochemical detection. The fibrosis related protein a-SMA and collagen I were assessed by Western blot. Different expressed genes (DEGs) between XYS-treated group and model group were analyzed. The herb-component-target network was constructed combined the network pharmacology. The predict targets and pathways were validated by in vitro and in vivo experiments. RESULTS: With XYS treatment, the liver function was significantly improved, and fibrotic changes were alleviated. The a-SMA and collagen I expression levels in the liver were also decreased in XYS-treated rats compared with CCl4 model rats. 108 active components and 42 targets from 8 herbs constituted herb-compound-target network by transcriptomics and network pharmacology analysis. The KEGG pathway and GO enrichment analyses showed that the FoxO, TGFß, AMPK, MAPK, PPAR, and hepatitis B and C pathways were involved in the anti-fibrosis effects of XYS. In the liver tissues, p-FoxO3a and p-Akt expression levels were significantly increased in the CCl4 model group but decreased in the XYS-treated group. The TGFß1/Smad pathway and Akt/FoxO3 pathway were verified in LX2 cells by inhibiting phosphorylation of Smad3 and Akt activity, respectively. CONCLUSIONS: Our findings suggested that XYS markedly alleviated CCl4-induced liver fibrosis in histopathological and serum liver function analyses, and this effect may occur via the TGFß1/Smad and Akt/FoxO signaling pathways.

Baihe Wuyao decoction ameliorates CCl4-induced chronic liver injury and liver fibrosis in mice through blocking TGF-ß1/Smad2/3 signaling, anti-inflammation and anti-oxidation effects.

ETHNOPHARMACOLOGICAL RELEVANCE: Baihe Wuyao decoction (BWD), a prescription of Traditional Chinese Medicines, composed of Lilium brownii var. viridulum Baker.(Lilii Bulbus) and Lindera aggregata (Sims) Kosterm. (Linderae Radix), has been used to treat epigastric pain and superficial gastritis for hundreds of years in China. Recently, some compounds obtained from Lilii Bulbus and Linderae Radix had active effects of hepatic protection or liver fibrosis alleviation. Thus, we aim to evaluate the effects of BWD on treatment of chronic liver injury and liver fibrosis induced by carbon tetrachloride (CCl4) and to elucidate the possible molecular mechanism. MATERIALS AND METHODS: Mice were treated with BWD (low, medium and high dose), diammonium glycyrrhizinate or vehicle by oral gavage once daily, simultaneously intraperitoneal injected with a single dose of CCl4 (1 µl/g body weight) twice a week for consecutive 6 weeks. Next, all mice were sacrificed after fasted 12 h, and serums and liver tissues were harvested for analysis. The hepatic injury was detected by serum biomarker assay, including aspartate aminotransferase (AST) and alanine aminotransferase (ALT). The hepatic histology and collagen were illustrated by hematoxylin-eosin staining and Sirius red staining respectively. The antioxidant capacity of liver tissues was evaluated by the contents of superoxide dismutase (SOD) and malondialdehyde (MDA) in liver homogenization. The mRNA gene or protein expressions related to fibrosis, oxidative stress and inflammation molecules were performed by real-time quantitative PCR (RT-PCR) or Western-blot. RESULTS: BWD exhibited a good hepatic protection with ameliorating liver histological changes, decreasing serum AST and ALT contents, and reducing hepatic fibrosis with stimulation ECMs (such as Collagen1 and Collagen3) degradation. BWD inhibited hepatic stellate cells (HSCs) activation, promoted matrix metalloproteinase-2 (MMP2), MMP9, and MMP12 while suppressing tissue inhibitors of matrix metalloproteinase-1 (TIMP1) expression, and blocked traditional fibrosis TGF-ß1/Smad2/3 signal pathway. Moreover, BWD exhibited anti-inflammation effect proved by the reduction of liver Interleukin-1ß (IL-1ß), TNF-α, IL-11 mRNA levels and promoted anti-oxidation effects determined by inhibition of liver MDA and iNOS levels while promoting liver SOD and Mn-SOD. CONCLUSION: BWD ameliorates CCl4-induced CLI and liver fibrosis which is correlated to its blocking TGF-ß1/Smad2/3 signaling, anti-inflammation, and anti-oxidation effects. BWD, as a small traditional prescription, is a promising treatment for CLI and liver fibrosis through multiple pharmacological targets.

In vitro screening for compounds from Hypericum longistylum with anti-pulmonary fibrosis activity.

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with a poor prognosis and limited therapies, and transforming growth factor-ß1 (TGF-ß1) plays a central role in the pathogenesis of IPF. Here, we aimed to investigate the chemical constituents and biological activities of Hypericum longistylum and detect whether the isolated compounds inhibit the TGF-ß1/Smad3 signaling pathway to identify candidate compounds for the treatment of pulmonary fibrosis. Fifteen compounds (1-15) were isolated from H. longistylum and their structures were elucidated on the basis of spectroscopic analyses. An in vitro MTT assay was used to test the effect of these fifteen compounds on fibroblast cytotoxicity and vitality. Furthermore, their bioactivities were screened using a TGF-ß1/Smad3 pathway luciferase reporter in vitro. MTT screening found that compounds 1-15 had no deleterious effects on normal mouse lung fibroblasts and no significant inhibition of vitality. Luciferase assay showed that compounds 14 and 15 could significantly inhibit the TGF-ß1/Smad3 pathway with the inhibition rates of 67.92% and 93.10%, respectively. Both compounds can be used as lead compounds for structural modification and optimization to obtain more drug candidates for the treatment of pulmonary fibrosis.

Combination of Ginsenoside Rg1 and Astragaloside IV reduces oxidative stress and inhibits TGF-ß1/Smads signaling cascade on renal fibrosis in rats with diabetic nephropathy.

INTRODUCTION: Anti-oxidative stress and inhibition of TGF-ß1/Smads signaling cascade are essential therapeutic strategies for diabetic nephropathy (DN). In this study, we aimed to explore the effect of combination of Ginsenoside Rg1 and Astragaloside IV on oxidative stress and TGF-ß1/Smads signaling in DN rats. MATERIALS AND METHODS: Wistar rats were divided into five groups: N group, M group (streptozotocin [STZ], intraperitoneally), G group (STZ rats with Ginsenoside Rg1, intragastrically [ig]), A group (STZ rats with Astragaloside IV, ig) and C group (STZ rats with Ginsenoside Rg1 and Astragaloside IV, ig). The levels of methane dicarboxylic aldehyde (MDA), catalase (CAT), glutathione peroxidase (GSH-PX), total anti-oxidative capacity (T-AOC), blood urea nitrogen (BUN), ß2-microglobulin (ß2-MG), serum creatinine (SCr) and urinary creatinine (UCr) were detected in all the groups. The left kidneys of the rats were harvested to detect the expression of TGF-ß1, Smad2/3, Smad7 and CTGF by immunohistochemical staining, while the right kidneys were used to detect the mRNA expression of TGF-ß1, Smad7 and CTGF by real-time PCR. RESULTS: Rats in G group, A group and C group had lower level of MDA but higher levels of CAT, GSH-PX and T-AOC compared with rats in M group. Rats in C group showed the best anti-oxidative stress level. G group, A group and C group treatments significantly decreased the levels of BUN, SCr, ß2-MG and UCr. In addition, C group treatment showed the best kidney protective effect. G group, A group and C group treatments significantly diminish ED both factor and mRNA overexpression of TGF-ß1 and CTGF but increase Smad7 expression in kidney tissue. CONCLUSION: The combination of Ginsenoside Rg1 and Astragaloside IV may potentially protect against DN by reducing oxidative stress and inhibiting TGF-ß1/Smads signaling cascade.

The preventive and therapeutic implication for renal fibrosis by targetting TGF-ß/Smad3 signaling.

It is well established that Smad3 is a key downstream effector of transforming growth factor-ß (TGF-ß) signaling in tissue fibrogenesis. We reported here that targetting Smad3 specifically with a Smad3 inhibitor SIS3 is able to prevent or halt the progression of renal fibrosis in a mouse model of unilateral ureteral obstructive nephropathy (UUO). We found that preventive treatment with SIS3 at the time of disease induction largely suppressed progressive renal fibrosis by inhibiting α-smooth muscle actin (α-SMA) + myofibroblast accumulation and extracellular matrix (collagen I (Col.I) and fibronectin (FN)) production. Importantly, we also found that treatment with SIS3 on established mouse model of UUO from day 4 after UUO nephropathy halted the progression of renal fibrosis. Mechanistically, the preventive and therapeutic effects of SIS3 on renal fibrosis were associated with the inactivation of Smad3 signaling and inhibition of TGF-ß1 expression in the UUO kidney. In conclusion, results from the present study suggest that targetting Smad3 may be a specific and effective therapy for renal fibrosis.

Branched chain amino acids supplementation modulates TGF-ß1/Smad signaling pathway and interleukins in CCl4 -induced liver fibrosis.

The alterations and low levels of circulating branched chain amino acids (BCAAs), leucine, isoleucine, and valine, are associated with liver diseases. The study was designed to evaluate hepatoprotective effect of BCAAs on CCl4 -induced liver fibrosis and to investigate the molecular mechanisms underlying these effects in rats. In all, 30 male rats were divided into three groups. Control group (n = 10) and CCl4 group (n = 10), where rats were injected with CCl4 (1 mL/kg of 0.5 : 1 v/v injected i.p. twice weekly for 12 weeks). In CCl4 + BCAAs group (n = 10), rats were injected with similar doses of CCl4 and supplemented with a mixture of 600 mg/kg BCAAs (2 : 1 : 1.2 leucine : isoleucine : valine) by oral gavage, three times/week for 12 weeks. Liver fibrosis was assessed by measuring total bilirubin, total protein, alanine aminotransferase, and aspartate aminotransferase, hydroxyproline content, and serum IL-6 and IL-10. Histopathologic studies and α-smooth muscle actin (α-SMA) were detected immunohistochemically in liver. Serum insulin level, blood glucose, liver malodialdehyde concentration (MDA), glutathione peroxidase, and superoxide dismutase (SOD) activities were quantified. TGF-ß1, Smad3, and Smad7 gene expressions were estimated by qRT-PCR. BCAAs suppressed liver fibrosis induced by CCl4 treatment. BCAAs modulated liver indices and downregulated TGF-ß1, Smad3, and Smad7 expressions in hepatocytes. BCAAs enhanced liver antioxidant enzyme activities (P < 0.001), reduced serum levels of TGF-ß1, IL-6, and IL-10 compared to CCL4 group and ameliorated histopathologic changes in rat liver. BCAAs may have a protective role against liver fibrosis via antioxidant and anti-inflammatory mechanisms.

Development of antisense oligonucleotide (ASO) technology against Tgf-ß signaling to prevent scarring during flexor tendon repair.

Flexor tendons (FT) in the hand provide near frictionless gliding to facilitate hand function. Upon injury and surgical repair, satisfactory healing is hampered by fibrous adhesions between the tendon and synovial sheath. In the present study we used antisense oligonucleotides (ASOs), specifically targeted to components of Tgf-ß signaling, including Tgf-ß1, Smad3 and Ctgf, to test the hypothesis that local delivery of ASOs and suppression of Tgf-ß1 signaling would enhance murine FT healing by suppressing adhesion formation while maintaining strength. ASOs were injected in to the FT repair site at 2, 6 and 12 days post-surgery. ASO treatment suppressed target gene expression through 21 days. Treatment with Tgf-ß1, Smad3 or Ctgf ASOs resulted in significant improvement in tendon gliding function at 14 and 21 days, relative to control. Consistent with a decrease in adhesions, Col3a1 expression was significantly decreased in Tgf-ß1, Smad3 and Ctgf ASO treated tendons relative to control. Smad3 ASO treatment enhanced the maximum load at failure of healing tendons at 14 days, relative to control. Taken together, these data support the use of ASO treatment to improve FT repair, and suggest that modulation of the Tgf-ß1 signaling pathway can reduce adhesions while maintaining the strength of the repair.

Chaihuang-Yishen granule inhibits diabetic kidney disease in rats through blocking TGF-ß/Smad3 signaling.

OBJECTIVE: Increasing evidence shows that TGF-ß1 is a key mediator in diabetic nephropathy (DN) and induces renal fibrosis positively by Smad3 but negatively by Smad7. However, treatment of DN by blocking the TGF-ß/Smad pathway remains limited. The present study investigated the anti-fibrotic effect of a traditional Chinese medicine, Chaihuang-Yishen granule (CHYS), on DN. RESEARCH DESIGN AND METHODS: Protective role of CHYS in DN was examined in an accelerated type 1 DN induced by streptozotocin in uninephrectomized Wistar rats. CHYS, at a dose of 0.56 g/kg body weight, was administered by a daily gastric gavage for 20 weeks and the therapeutic effect and potential mechanisms of CHYS on diabetic kidney injury were examined. RESULTS: Treatment with CHYS attenuated diabetic kidney injury by significantly inhibiting 24-h proteinuria and progressive renal fibrosis including glomerulosclerotic index, tubulointerstitial fibrosis index, and upregulation of extracellular matrix (collagen I, IV, and fibronectin), despite the same levels of blood glucose. Further studies revealed that inhibition of renal fibrosis in CHYS-treated diabetic rats was associated with inhibition of TGF-ß1/Smad3 signaling as demonstrated by upregulation of Smad7 but downregulation of TGF-ß1, TGF-ß receptors, activation of Smad3, and expression of miRNA-21. CONCLUSIONS: CHYS may be a therapeutic agent for DN. CHYS attenuates DN by blocking TGF-ß/Smad3-mediated renal fibrosis.

Gualou Xiebai Decoction prevents myocardial fibrosis by blocking TGF-beta/Smad signalling.

OBJECTIVES: The present study is aimed to investigate the effect of Gualou Xiebai Decoction (GXD) ethanol extract on myocardial fibrosis and clarify the possible mechanism. METHODS: Rats with ligated left anterior descending coronary artery were treated with GXD ethanol extract (1.14 g/kg, 2.27 g/kg, 4.53 g/kg) daily via gavage for 4 weeks. Histopathological changes and collagen distribution were evaluated by haematoxylin and eosin and Masson staining. The mRNA levels of Collagen I and Collagen III were detected by real-time PCR. The expressions of TGF-ß1, TGFß receptor (TGFßR)I, TGFßRII, P-Smad2/3 and Smad7 were determined by Western blot. RESULTS: GXD treatment was significantly reduced the heart weight/body weight ratio (P < 0.05) as well as the left ventricle weight/body weight ratio (P < 0.05). It also significantly alleviated the degree of inflammation, decreased myocardial collagen volume fraction (P < 0.05 ∼ 0.01), together with markedly prevented the upregulations of Collagen I and Collagen III (P < 0.05 ∼ 0.01). Moreover, GXD downregulated expressions of TGF-ß1, TGFßRI, TGFßRII, Smad2/3 whereas improved Smad7 expression in the myocardial fibrosis rats. CONCLUSIONS: GXD ameliorates myocardial fibrosis induced by cardiac infarction with ligated left anterior descending coronary artery, the mechanism maybe involve in inhibiting the TGF-ß1 signalling pathway.

Inhibition of miR-29 by TGF-beta-Smad3 signaling through dual mechanisms promotes transdifferentiation of mouse myoblasts into myofibroblasts.

MicroRNAs (miRNAs) are non-coding RNAs that regulate gene expression in post-transcriptional fashion, and emerging studies support their importance in regulating many biological processes, including myogenic differentiation and muscle development. miR-29 is a promoting factor during myogenesis but its full spectrum of impact on muscle cells has yet to be explored. Here we describe an analysis of miR-29 affected transcriptome in C2C12 muscle cells using a high throughput RNA-sequencing platform. The results reveal that miR-29 not only functions to promote myogenic differentiation but also suppresses the transdifferentiation of myoblasts into myofibroblasts. miR-29 inhibits the fibrogenic differentiation through down-regulating both extracellular matrix genes and cell adhesion genes. We further demonstrate that miR-29 is under negative regulation by TGF-beta (TGF-ß)-Smad3 signaling via dual mechanisms of both inhibiting MyoD binding and enhancing Yin Yang 1 (YY1)-recruited Polycomb association. Together, these results identify miR-29 as a pleiotropic molecule in both myogenic and fibrogenic differentiation of muscle cells.

Scutellaria extract and wogonin inhibit tumor-mediated induction of T(reg) cells via inhibition of TGF-ß1 activity.

A number of studies have implicated tumor-induced T(reg) cell activity in the sub-optimal response to therapeutic vaccines. Development of neo-adjuvant strategies targeting T(reg) cells is therefore imperative. Scutellaria extracts or constituent flavonoids have shown encouraging efficacy against various tumors, including gliomas, in both pre-clinical and clinical studies. We report here, for the first time, that Scutellaria ocmulgee leaf extract (SocL) and flavonoid wogonin could inhibit TGF-ß1-induced T(reg) activity in malignant gliomas. F344 rats, subcutaneously transplanted with F98 gliomas, were treated with SocL. There was a significant inhibition of intra-tumoral TGF-ß1 and T(reg) cell frequency as well as peripheral blood TGF-ß1 levels in SocL-treated animals compared to the controls. SocL extract and wogonin also inhibited glioma-induced, TGF-ß1-mediated T(reg) activity in vitro. SocL extract and wogonin also inhibited the secretion of IL-10 in T(reg) culture; whereas the level of IL-2 was either unchanged or marginally enhanced. We also observed an inhibition of Smad-3, GSK-3ß and ERK1/2 signaling by SocL and wogonin in T(reg) cells, while phosphorylation of P38 MAPK was considerably enhanced, indicating that SocL or wogonin could inhibit the T cells' response to TGF-ß1 via modulation of both Smad and non-Smad signaling pathways. Overall, this study suggests that Scutellaria can potentially reverse tumor-mediated immune suppression via inhibition of TGF-ß1 secretion as well as via inhibition of T cells' response to TGF-ß1. This may provide an opportunity for developing a novel adjuvant therapeutic strategy for malignant gliomas, combining Scutellaria with immunotherapy and chemo/radio-therapeutic regimen, which could potentially improve the disease outcome.

Schisandrin B suppresses TGFß1 signaling by inhibiting Smad2/3 and MAPK pathways.

TGFß1 plays a crucial role in the pathogenesis of vascular fibrotic diseases. Schisandra chinensis (S. chinensis), which is used as an oriental herbal medicine, is effective in the treatment of vascular injuries that cause aberrant TGFß1 signaling. In this study, we investigated whether S. chinensis extract and its active ingredients inhibit TGFß1 signaling in A7r5 vascular smooth muscle cells. We found that S. chinensis extract suppressed TGFß1 signaling via inhibition of Smad2/3 phosphorylation and nuclear translocation. Among the active ingredients of S. chinensis extract, schisandrin B (SchB) most potently inhibited TGFß1 signaling. SchB inhibited sustained phosphorylation and nuclear translocation of Smad2/3. Moreover, SchB suppressed TGFß1-induced phosphorylation of p38 and JNK, which contributed to Smad2/3 inactivation. The present study is the first to demonstrate that S. chinensis extract and SchB inhibit TGFß1 signaling. Our results may help future investigations to understand vascular fibrosis pathogenesis and to develop novel therapeutic strategies for treatment of vascular fibrotic diseases.

Inhibition of arsenic-induced rat liver injury by grape seed exact through suppression of NADPH oxidase and TGF-ß/Smad activation.

Chronic arsenic exposure induces oxidative damage to liver leading to liver fibrosis. We aimed to define the effect of grape seed extract (GSE), an antioxidant dietary supplement, on arsenic-induced liver injury. First, Male Sprague-Dawley rats were exposed to a low level of arsenic in drinking water (30ppm) with or without GSE (100mg/kg, every other day by oral gavage) for 12months and the effect of GSE on arsenic-induced hepatotoxicity was examined. The results from this study revealed that GSE co-treatment significantly attenuated arsenic-induced low antioxidant defense, oxidative damage, proinflammatory cytokines and fibrogenic genes. Moreover, GSE reduced arsenic-stimulated Smad2/3 phosphorylation and protein levels of NADPH oxidase subunits (Nox2, Nox4 and p47phox). Next, we explored the molecular mechanisms underlying GSE inhibition of arsenic toxicity using cultured rat hepatic stellate cells (HSCs). From the in vitro study, we found that GSE dose-dependently reduced arsenic-stimulated ROS production and NADPH oxidase activities. Both NADPH oxidases flavoprotein inhibitor DPI and Nox4 siRNA blocked arsenic-induced ROS production, whereas Nox4 overexpression suppressed the inhibitory effects of GSE on arsenic-induced ROS production and NADPH oxidase activities, as well as expression of TGF-ß1, type I procollagen (Coll-I) and α-smooth muscle actin (α-SMA) mRNA. We also observed that GSE dose-dependently inhibited TGF-ß1-induced transactivation of the TGF-ß-induced smad response element p3TP-Lux, and that forced expression of Smad3 attenuated the inhibitory effects of GSE on TGF-ß1-induced mRNA expression of Coll-I and α-SMA. Collectively, GSE could be a potential dietary therapeutic agent for arsenic-induced liver injury through suppression of NADPH oxidase and TGF-ß/Smad activation.