Kallistatin protects against bleomycin-induced idiopathic pulmonary fibrosis by inhibiting angiogenesis and inflammation.

Aberrant angiogenesis and vascular remodeling are the main features of idiopathic pulmonary fibrosis. Kallistatin is an anti-angiogenic peptide with known effects on endothelial cells. This study aimed to demonstrate that kallistatin has beneficial effects on bleomycin (BLM)-induced pulmonary fibrosis in a rat model by inhibiting angiogenesis. Twenty-five rats were randomly divided into five experimental groups: (A) Saline only (SA)-as the negative control, (B) BLM only (BLM)-as the model group, (C) BLM and 0.1 mg/kg kallistatin (L-Kal), (D) BLM and 0.5 mg/kg kallistatin (M-Kal), and (E) BLM and 2.5 mg/kg kallistatin (H-Kal). Fibrillar collagen was quantified by Masson's trichrome and hematoxylin-eosin staining. Transforming growth factor-ß1 (TGF-ß1), α-smooth-muscle-actin (α-SMA) and microvascular density (MVD) were measured by immunohistochemistry. Vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor (VEGFR), and tumor necrosis factor-α (TNF-α) were assayed by Western immunoblotting or ELISA. Daily administration of kallistatin attenuated fibrosis in BLM-induced pulmonary fibrosis, as shown by histology. During inflammation from BLM-induced pulmonary fibrosis, kallistatin reduced the number of inflammatory cells infiltrating the bronchoalveolar lavage fluid. Kallistatin also inhibited VEGF expression and phosphorylation of VEGFR2 (Flk-1). In vitro, kallistatin blocked tube formation by inhibiting Flk-1 and GSK-3ß phosphorylation. The results demonstrated that continuous administration of kallistatin attenuated BLM-induced pulmonary fibrosis and improved survival of BLM rats. Reducing pulmonary fibrosis was achieved by partial inhibition of pulmonary inflammation and angiogenesis.

Achyranthes bidentate saponins protect rat articular chondrocytes against interleukin-1ß-induced inflammation and apoptosis in vitro.

Achyranthes bidentate Blume (Niuxi) is often employed for treatment of arthritis in Traditional Chinese Medicine and possesses anti-inflammatory properties. Phytochemical and pharmacological studies proved the oleanane-type saponins to be the main bioactive principles. In the present study, protective effects of A. bidentata saponins (ABS) on inflammation and apoptosis in interleukine-1ß (IL-1ß)-induced chondrocytes were investigated. Rat chondrocytes were pretreated with ABS at 3 µg/mL, 10 µg/mL, and 30 µg/mL, and subsequently stimulated with IL-1ß (10 ng/mL). Methylthiazolyldiphenyl-tetrazolium bromide assay and annexin V/propidium iodide dual staining demonstrated that ABS could protect IL-1ß-induced chondrocyte injury. ABS suppressed IL-1ß-induced apoptosis by suppressing the activation of caspase-3, inhibiting levels of proapoptotic proteins Bax and Bad, decreasing p53 protein phosphorylation, and promoting the expression of antiapoptotic protein Bcl-xL and proliferating cell nuclear antigen. IL-1ß-induced inflammation and matrix degradation were also alleviated by ABS through the downregulation of the expressions of matrix metalloproteinases 3 and 9 and cyclooxygenase-2. Moreover, ABS inhibited IL-1ß-induced nuclear factor κB activation in rat chondrocytes. We demonstrated, for the first time, the protective effects of ABS on IL-1ß-stimulated chondrocytes and their molecular mechanisms. Thus, it is suggested that ABS might be a potential drug in the treatment of osteoarthritis.

Uric acid transporters hiding in the intestine.

CONTEXT: Hyperuricaemia is known as an abnormally increased uric acid level in the blood. Although it was observed many years ago, since uric acid excretion via the intestine pathway accounted for approximately one-third of total elimination of uric acid, the molecular mechanism of 'extra-renal excretion' was poorly understood until the finding of uric acid transporters. OBJECTIVE: The objective of this study was to gather all information related to uric acid transporters in the intestine and present this information as a comprehensive and systematic review article. METHODS: A literature search was performed from various databases (e.g., Medline, Science Direct, Springer Link, etc.). The key terms included uric acid, transporter and intestine. The period for the search is from the 1950s to the present. The bibliographies of papers relating to the review subject were also searched for further relevant references. RESULTS: The uric acid transporters identified in the intestine are discussed in this review. The solute carrier (SLC) transporters include GLUT9, MCT9, NPT4, NPT homolog (NPT5) and OAT10. The ATP binding cassette (ABC) transporters include ABCG2 (BCRP), MRP2 and MRP4. Bacterial transporter YgfU is a low-affinity and high-capacity transporter for uric acid. CONCLUSION: The present review may be helpful for further our understanding of hyperuricaemia and be of value in designing future studies on novel therapeutic pathways.

Access to Indole Derivatives from Diaryliodonium Salts and 2-Alkynylanilines.

An efficient, environmentally friendly, and operationally simple procedure to 1,2-disubstituted indoles from 2-alkynylanilines and diaryliodonium salts has been developed. This reaction proceeds smoothly under metal-free conditions. The products obtained could be transferred into 3,3'-diindolylmethane with DMSO catalyzed by palladium. The isotopic label experiments indicated that the methylene group in 3,3'-diindolylmethane is derived from DMSO. The diverse indoles were obtained in up to 90% yield for 28 examples.

[The effect of Connexin43 downregulation on biological functions of HUVEC].

Connexin43 has been shown to play a pivotal role in wound healing process. Wound repair is enhanced by acute downregulation of connexin43, by increasing proliferation and migration of keratinocyte and fibroblast. Angiogenesis is also a central feature of wound repair, but little is known about the effects of connexin43 modulation on functions of endothelial cells. We used connexin43 specific small interference RNA (siRNA) to reduce the expression of connexin43 in human umbilical vein endothelial cell (HUVEC), and investigated the effects of connexin43 downregulation on intercellular communication, viability, proliferation, migration and angiogenic activity of HUVEC. Treatment of siRNA markedly reduced the expression of connexin43 by -80% in HUVEC (P < 0.05), and decreased the intercellular communication by -65% (P < 0.05). The viability, proliferation, migration and angiogenic activity of HUVEC decreased significantly (P < 0.05), compared with that of the normal cells. The results suggest that temporally downregulation of connexin43 expression at early stage of wound to inhibit the abnormal angiogenesis characterized with leaky and inflamed blood vessels, maybe a prerequisite for coordinated normal healing process.

Ginsenoside Ro suppresses interleukin-1ß-induced apoptosis and inflammation in rat chondrocytes by inhibiting NF-κB.

This study investigated effects of Ginsenoside Ro (Ro) on interleukin-1ß (IL-1ß)-induced apoptosis and inflammation in rat chondrocytes. The rat chondrocytes were co-treated with IL-1ß (10 ng·kg(-1)) and Ro (50, 100 and 200 µmol·L(-1)) for 48 h. Chondrocytes viability was detected by the MTT assay and Annexin V-FITC/PI dual staining assay. Caspase 3 activity was measured by using caspase 3 colorimetric assay kit. Apoptosis related proteins Bax, Bad, Bcl-xL, PCNA, p53 and phospho-p53, along with inflammation related protein MMP 3, MMP 9 and COX-2, and the expression of phospho-NF-κB p65 were assayed by western blotting analyses. Ro could improve IL-1ß-induced chondrocytes viability. Ro could suppress IL-1ß-induced apoptosis by inhibiting levels of Bax and Bad, decreasing p53 phosphorylation and promoting the expression of Bcl-xL and PCNA. Ro inhibited caspase 3 activity. IL-1ß-induced inflammation and matrix degration were also alleviated by Ro with down-regulating the expression of MMP 3, MMP 9 and COX-2. Moreover, Ro inhibited NF-κB p65 phosphorylation induced by IL-1ß. In conclusion, these results suggested Ro exerted anti-apoptosis and anti-inflammation in IL-1ß-induced rat chondrocytes, which might be related to NF-κB signal pathway. Therefore, we propose that Ro might be a potential novel drug for the treatment of osteoarthritis.

Neuroprotection by saponins.

Saponins, an important group of bioactive plant natural products, are glycosides of triterpenoid or steroidal aglycones. Their diverse biological activities are ascribed to their different structures. Saponins have long been recognized as key ingredients in traditional Chinese medicine. Accumulated evidence suggests that saponins have significant neuroprotective effects on attenuation of central nervous system disorders, such as stroke, Alzheimer's disease, Parkinson's disease, and Huntington's disease. However, our understanding of the mechanisms underlying the observed effects remains incomplete. Based on recently reported data from basic and clinical studies, this review highlights the proposed mechanisms of their neuroprotective function including antioxidant, modulation of neurotransmitters, anti-apoptosis, anti-inflammation, attenuating Ca(2+) influx, modulating neurotrophic factors, inhibiting tau phosphorylation, and regeneration of neural networks.

Copper-catalyzed one-pot synthesis of unsymmetrical arylurea derivatives via tandem reaction of diaryliodonium salts with N-arylcyanamide.

An efficient "one-pot" approach to multiple substituted ureas from N-arylcyanamide and diaryliodonium salts has been presented. The two-step procedure involved the weak base-promoted chemoselective arylation of secondary amines with diaryliodonium and Cu-catalyzed nucleophilic addition of N-arylcyanamide with second diaryliodonium. The diverse unsymmetrical arylureas were obtained in up to 91% yield for 29 examples.

ß-Ecdysterone suppresses interleukin-1ß-induced apoptosis and inflammation in rat chondrocytes via inhibition of NF-κB signaling pathway.

Osteoarthritis (OA) is characterized by a loss of articular cartilage accompanied with inflammation of synovium. ß-Ecdysterone (Ecd), a major component of several Chinese herbal medicines, e.g., Achyranthes bidentata BL., has been used for the prevention and treatment of OA. Ecd is an estrogen analog and is likely to have similar pharmacological effects including the effect of protective chondrocytes. This study investigated the effects of Ecd on interleukin-1ß (IL-1ß)-induced apoptosis and inflammation in rat chondrocytes. Ecd protected chondrocytes from IL-1ß-induced injury by inhibiting expression of Bax, p53 phosphorylation, and promoting expression of Bcl-xL . Simultaneously, Ecd reduced caspase 3 activity. IL-1ß-induced inflammation and matrix degration were also prevented by Ecd via down-regulation of matrix metalloproteinases MMP 3, MMP 9, and cyclooxygenase-2 expression. Additionally, Ecd inhibited Nuclear Factor Kappa B (NF-κB) p65 phosphorylation, IκBα degradation, and phosphorylation in IL-1ß-induced rat chondrocytes. These results suggested Ecd exerted anti-apoptosis and anti-inflammation in IL-1ß-induced rat chondrocytes, which might be related to NF-κB signal pathway.

Saponin-rich fraction from Clematis chinensis Osbeck roots protects rabbit chondrocytes against nitric oxide-induced apoptosis via preventing mitochondria impairment and caspase-3 activation.

Our previous study reported that the saponin-rich fraction from Clematis chinensis Osbeck roots (SFC) could effectively alleviate experimental osteoarthritis induced by monosodium iodoacetate in rats through protecting articular cartilage and inhibiting local inflammation. The present study was performed to investigate the preventive effects of SFC on articular chondrocyte, and explore the underlying mechanisms. Primary rabbit chondrocytes were cultured and exposed to sodium nitroprusside (SNP), a NO donor. After treatment with different concentrations of SFC (30, 100, 300, 1,000 µg/ml) for 24 h, nucleic morphology, apoptotic rate, mitochondrial function and caspase-3 activity of chondrocytes were examined. The results showed that SNP induced remarkable apoptosis of rabbit chondrocytes evidenced by Hoechst 33258 staining and flow cytometry analysis, and SFC prevented the apoptosis in a concentration-dependent manner. Further studies indicated that SFC could prevent the depolarization of mitochondrial membrane potential (∆ψm) in SNP-treated chondrocytes and suppress the activation of caspase-3. It can be concluded that the protection of SFC on articular chondrocytes is associated with the anti-apoptosis effects via inhibiting the mitochondrion impairment and caspase-3 activation.

Paeoniflorin attenuates lipopolysaccharide-induced permeability of endothelial cells: involvements of F-actin expression and phosphorylations of PI3K/Akt and PKC.

This study aimed to investigate the effects of paeoniflorin, the main active ingredient of the medicinal plant Paeonia lactiflora Pall., on the permeability of endothelial cells induced by lipopolysaccharide (LPS) and the underlying mechanisms. Human umbilical vein endothelial cells (HUVECs) were stimulated by LPS. Extravasated FITC-dextran reflecting permeability was assessed by multimode microplate reader, and the migration of bis-carboxyethyl-carboxyfluorescein acetoxy-methyl-labeled human acute monocytic leukemia cell line and leukemia cell line cells through HUVECs were analyzed by fluorescence microscopy. The phosphorylations of phosphatidylinositol 3-kinase (PI3K)/Akt, protein kinase C (PKC), and cofilin in HUVECs were assessed by western blotting, and the F-actin level was detected by laser scanning confocal microscopy. After LPS stimulation, inflammatory endothelial cells exhibited significantly increased permeability. Paeoniflorin (10, 30, and 100 µM) inhibited dextran extravasation and leukocyte migration through HUVECs induced by LPS in a concentration-dependent manner. Moreover, paeoniflorin was able to suppress the phosphorylations of PI3K/Akt, PKC, and cofilin, as well as F-actin reorganization in HUVECs induced by LPS. These findings revealed that paeoniflorin partly blocked LPS-induced endothelium permeability, supporting a new explanation for its anti-inflammatory effects.

[Research progresses of plant polysaccharides on cell adhesion].

Cell adhesion mediated by cell adhesion molecules (CAMs) constitutes essential life phenomenon. In inflammation, immunity, infection, thrombosis, tumor metastasis and wound healing, cell adhesion comes into being the basic physiological and pathological process. Intervening with cell adhesion has been the important therapeutic and prophylactic strategies for diseases. Accumulated evidence has indicated that plant polysaccharides especially those exacted from Chinese traditional and herbal drugs displayed various pharmacological effects such as anti-inflammation, anti-cancer, anti-infection, immunomodulation, cardiovascular protective effects and so on. In this paper, the research progress of plant polysaccharides on cell adhesion is reviewed.

Prevention of FGF-2-induced angiogenesis by scopoletin, a coumarin compound isolated from Erycibe obtusifolia Benth, and its mechanism of action.

Previous work in our laboratory has shown that scopoletin, one of the main bioactive constituents of Erycibe obtusifolia Benth stems, exerts anti-arthritic activity in vivo partly by preventing synovial angiogenesis. The present study was performed to further investigate the anti-angiogenic potential of scopoletin, focusing on the mechanisms of action in vitro. In the aortic ring sprouting assay, scopoletin (10, 30 and 100 µM) significantly inhibited the growth of endothelial sprouts in a concentration-dependent manner. As to human umbilical vein endothelial cells (HUVECs), scopoletin could inhibit their proliferation, migration and tubule formation induced by FGF-2, especially the proliferation. It also remarkably decreased the expression of VEGF at mRNA and protein levels, and the phosphorylations of IKKα and IκB but not Akt, as well as the degradation of IκB caused by FGF-2 in HUVECs. These findings suggest that scopoletin is substantially able to attenuate FGF-2-induced angiogenesis, and it might act by directly preventing the stimulation action of FGF-2 and by indirectly decreasing the production of VEGF. Scopoletin down-regulated the VEGF expression through NF-κB rather than PI-3K/Akt signaling pathway.

Polysaccharide of radix pseudostellariae improves chronic fatigue syndrome induced by poly I:C in mice.

Radix Pseudostellariae is used as a tonic drug in traditional Chinese medicine with immunomodulating and anti-fatigue activities, and the polysaccharide is considered as the main active component. The purpose of this study is to examine the effect of the polysaccharide isolated from Radix Pseudostellariae (PRP) on mouse chronic fatigue syndrome (CFS) induced by intraperitoneal injection of polyriboinosinic:polyribocytidylic acid (poly I:C), a double-stranded synthetic RNA. It has shown that the fatigue symptom of mice lasted at least 1 week as evaluated by forced swimming time. PRP (100, 200, 400 mg kg(-1)), orally administered 3 days before poly I:C injection, showed dose-dependent anti-fatigue effects. In addition, poly I:C led to evident alternations in neuroendocrine and immune systems of mice, such as reduced spontaneous activity and learning ability, declined serum level of corticosterone, increased weight indexes and T lymphocyte numbers in thymuses and spleens, and increased CD4(+)/CD8(+) ratio but decreased proliferation ability of T lymphocytes in spleens. PRP alleviated the abnormalities caused by poly I:C, and restored the function of hosts to normal conditions. The findings suggest that PRP is beneficial to CFS, and the underlying mechanisms of action involve neuroendocrine and immune systems.

Qi-Shao-Shuang-Gan, a combination of Astragalus membranaceus saponins with Paeonia lactiflora glycosides, ameliorates polymicrobial sepsis induced by cecal ligation and puncture in mice.

The present study was performed to investigate the anti-septic effects of Qi-Shao-Shuang-Gan (QSSG), a combination of Astragalus membranaceus saponins (SAM) and Paeonia lactiflora glycosides (GPL), in septic mice induced by cecal ligation and puncture. QSSG was shown to elevate the survival rate of mice, decrease infiltration of polymorphonuclear leukocytes into livers and lungs, lower serum levels of myeloperoxidase, nitric oxide, and lactate dehydrogenase, and decrease mRNA expressions of inducible nitric oxide synthase and interleukin-1ß in livers. It also restored the impaired expressions of protein C (PC) mRNA in mouse livers and expressions of thrombomodulin and endothelial PC receptor mRNA in endothelial cells. Neither SAM nor GPL alone could significantly increase the survival rate of septic mice. The findings indicate that QSSG exerts protective action against polymicrobial sepsis by inhibiting systemic inflammatory response and upregulating PC pathway, and there are synergistic effects between SAM and GPL.

A simple adhesion assay for studying interactions between platelets and endothelial cells in vitro.

Cell adhesion plays a key role during various physiological and pathological processes. Many studies have been performed to understand the interaction of platelets with endothelial cells (ECs) during the past decades. Modulation of their interaction has been shown to be therapeutically useful in thrombotic diseases. Some methods of labeling platelets such as counting and radiolabeling have been applied in the study of the platelets-ECs interaction, but these methods did not obtain full approval. A rapid, simple and sensitive assay for platelets-ECs interaction was developed in this paper. Platelets were labeled with Sudan Black B (SBB) before adding to confluent ECs monolayer. Non-adherent platelets were removed by washing with PBS. The adherent platelets were lysed with dimethylsulfoxide (DMSO) and the absorbance was recorded at 595 nm by spectrophotometer. A linear correlation was observed between the absorbance of SBB and the number of platelets. By employing the SBB method, the influence of heparin on platelets-ECs interactions was observed. Heparin (3-100 units/mL) obviously reduced platelets adhering to ECs in a concentration-dependent manner.

Therapeutic effect of the saponin fraction from Clematis chinensis Osbeck roots on osteoarthritis induced by monosodium iodoacetate through protecting articular cartilage.

The objective of the present study was to investigate the effect of the saponin fraction from Clematis chinensis Osbeck roots (SFC) on an osteoarthritis model in rats and to explore its underlying mechanisms. Osteoarthritis was induced by intraarticular injection of monosodium iodoacetate (MIA) into knee joints of rats, and SFC and diclofenac were orally administered once a day for 28 consecutive days. Joint swelling, macroscopic observation, histological assessment and proteoglycan (PG) degradation were examined. In vitro, cultured rabbit chondrocytes were stimulated with MIA and sodium nitroprusside (SNP), respectively. The effects of SFC on MIA- and SNP-induced chondrocyte injury were examined by MTT assay. It was shown that SFC (50, 100, 200 mg/kg) dose-dependently reduced cartilage injury and PG degradation induced by MIA. Diclofenac (4 mg/kg) only slightly alleviated cartilage injury and PG degradation. SFC also prevented SNP- or MIA-induced rabbit chondrocyte impairment. These results indicate that SFC is effective in ameliorating joint destruction and cartilage erosion in MIA-induced osteoarthritic in rats, and the mechanisms of action for protecting articular cartilage are through preventing extracellular matrix degradation and chondrocyte injury.

Heparin: an intervenor in cell communication.

It was nearly 100 years since heparin was discovered, but the role of this widely used anticoagulant is still remarkably thought provoking now. During pathological processes such as atherosclerosis, inflammation, cancer and infection, phenomena of cell adhesion are ubiquitous and complicated. Heparin exerts anti-adhesion activity appearing as a common mechanism of its potential polypharmacology in those diseases. Furthermore, heparin can bind a variety of signalling molecules such as growth factors, cell surface proteins of pathogens and most notably, cell adhesion molecules. These signalling molecules are involved in cell communication, acting as ligands, receptors and second messengers. Considering that heparan sulphate glycosaminoglycan is increasingly recognized as a key mediator in many cellular processes, the structural similarity with heparan sulphate suggests that heparin is a multifunctional intervenor in cell communication.

Saponin fraction from Astragalus membranaceus roots protects mice against polymicrobial sepsis induced by cecal ligation and puncture by inhibiting inflammation and upregulating protein C pathway.

Sepsis remains the leading cause of death in intensive care units. Uncontrolled systemic inflammation and an impaired protein C pathway are two important contributors to sepsis pathophysiology. Based on the beneficial effects of the saponin fraction from Astragalus membranaceus roots (SAM) against inflammation, liver dysfunction, and endothelium injury, we investigated the potential protective roles and underlying mechanisms of SAM on polymicrobial sepsis induced by cecal ligation and puncture (CLP) in mice. SAM, orally administered 1 h before and after CLP, significantly elevated the survival rate of mice. At 96 h after CLP operation, all mice in the model group died, whereas 33.3% of mice in the SAM (400 mg/kg)-treated group survived. SAM attenuated both inflammatory factors and their abilities to induce tissue dysfunction, which was mainly evidenced by decreased infiltration of polymorphonuclear leukocytes, tissue edema, and lung wet-to-dry weight ratio, lowered levels of myeloperoxidase (MPO), nitric oxide (NO), lactate dehydrogenase (LDH), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) in serum, as well as downregulated expressions of iNOS and IL-1beta mRNA in livers. Furthermore, we addressed the effects of SAM on the protein C (PC) pathway, closely linked with sepsis. In CLP-induced septic mice, SAM elevated the impaired expression of PC mRNA in livers. In vitro, SAM reversed the decreased expressions of thrombomodulin (TM) and endothelial PC receptor (EPCR) mRNA induced by lipopolysaccharide (LPS) in endothelial cells. These findings suggest that SAM is able to restore the impaired protein C pathway. Taken together, the current study demonstrates that SAM has protective effects on polymicrobial sepsis in mice. The mechanisms of action involve anti-inflammation and upregulation of the PC pathway.

[Research on effect of total saponins of yinfenglun on uterine].

OBJECTIVE: To research the effect of total saponins of Yinfenglun on uterine. METHOD: Models of uterine inflammation were established to observe the effect of total saponins of Yinfenglun. Uterine contractive effects were studied on rats in vitro and on rabbit in vivo. Weight of uterus and levels of estrogen and progestogen were determined. RESULT: Total Saponins of Yinfenglun had the ameliorated tendency to metritis of model rats, and increased the contractive range and motorricity of uterine of rats in vitro and of rabbit in vivo. The effect to uterus in vivo maintained longer. Total saponins of Yinfenglun could increase the weight of uterus and have an increased tendency on the content of estrogen, but not the level of progestogen. CONCLUSION: There are obvious effects on uterine of total saponins of Yinfenglun, which are related to its clinical use.