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.

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.

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.

A novel Gallic acid derivative attenuates BLM-induced pulmonary fibrosis in mice.

Idiopathic Pulmonary fibrosis is a disease with high morbidity and mortality. Therefore, the development of new drugs is imperative. Gallic acid derivative is a derivative of Gallic acid that can be extracted from Chinese herbal medicine. In previous experiments, we found that Gallic acid derivative played dual roles in inflammatory and antioxidant activities. Meanwhile, Gallic acid derivative could inhibit the proliferation of lung fibroblast. In the present study, we investigated the function of Gallic acid derivative in inhibiting lung fibrosis. 5 mg/kg of bleomycin was administered to mice by a single intratracheal instillation. Three dosages of Gallic acid derivative (75 mg/kg, 150 mg/kg, 300 mg/kg) and Pirfenidone (80 mg/kg) were given to mice for 21 day. Gallic acid derivative treatment significantly reduced lung histological changes and decreased inflammatory cell infiltration. The content of collagen decreased with the decrease of hydroxyproline level. Analogously, the expression of alpha smooth muscle actin was reduced. Gallic acid derivative enhanced the antioxidant status, but reduced the expression of interleukin 6, NADPH oxidase-4. Our study proved that Gallic acid derivative reduced inflammation activation to some extent and could exert its effects through transforming growth factor ß1/Smad2 signaling pathway and balancing NOX4/Nrf2.

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.

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.

Compound Astragalus and Salvia miltiorrhiza Extract exerts anti-fibrosis by mediating TGF-beta/Smad signaling in myofibroblasts.

Previous studies showed that Compound Astragalus and Salvia miltiorrhiza Extract (CASE) has a protective effect against liver fibrosis. We hypothesized that CASE exerts the anti-fibrosis effect by mediating transforming growth factor-beta (TGF-beta)/Smad signaling pathway. To test this hypothesis, we induced fibrosis in rats by twice weekly injections of carbon tetrachloride (CCl(4)) and Smad2 phosphorylation was measured by immunohistochemical method; protein expression in myofibroblasts (MFBs) induced by TGF-beta1 was analyzed by western blotting and plasminogen activator inhibitor type 1 (PAI-1) transcriptional activity in MFBs was evaluated. The present study showed that, in vivo, CASE has protective effects against liver fibrosis in rats generated by CCl(4), and that CASE inhibits Smad2 phosphorylation at C-terminal region and expression of alpha-smooth muscle actin (alpha-SMA). Our experiment further demonstrated that, in vitro, (1) CASE inhibits TGF-beta(1)-dependent Smad2 phosphorylation at C-terminal region and Smad2 and Smad3 phosphorylation at linker region in MFBs in a dose-dependent manner; (2) CASE decreases the level of Smad 2/3/4 complex in MFBs induced by TGF-beta(1) in a dose-dependent manner; (3) CASE inhibits PAI-1 transcriptional activity in MFBs induced by TGF-beta(1) in a dose-dependent manner; and (4) CASE markedly decreases c-Jun N-terminal kinase (JNK) phosphorylation in MFBs induced by TGF-beta(1). Our results suggest that CASE's anti-fibrosis effect in chronically injured liver was exerted by inhibiting TGF-beta/Smads signal transduction.