In the present study, we investigated anti-inflammatory effect of Cardamine komarovii flower (CKF) on lipopolysaccharide (LPS)-induced acute lung injury (ALI). We determined the effect of CKF methanolic extracts on LPS-induced pro-inflammatory mediators NO and prostaglandin E2 (PGE2), production of pro-inflammatory cytokines (IL-1β, TNF-α, and IL-6), and related protein expression levels of MyD88/TRIF signaling pathways in peritoneal macrophages (PMs). Nuclear translocation of NF-κB-p65 was analyzed by immunofluorescence. For the in vivo experiments, an ALI model was established to detect the number of inflammatory cells and inflammatory factors (IL-1β, TNF-α, and IL-6) in bronchoalveolar lavage fluid (BALF) of mice. The pathological damage in lung tissues was evaluated through H&E staining. Our results showed that CKF can decrease the production of inflammatory mediators, such as NO and PGE2, by inhibiting their synthesis-related enzymes iNOS and COX-2 in LPS-induced PMs. In addition, CKF can downregulate the mRNA levels of IL-1β, TNF-α, and IL-6 to inhibit the production of inflammatory factors. Mechanism studies indicated that CKF possesses a fine anti-inflammatory effect by regulating MyD88/TRIF dependent signaling pathways. Immunocytochemistry staining showed that the CKF extract attenuates the LPS-induced translocation of NF-kB p65 subunit in the nucleus from the cytoplasm. In vivo experiments revealed that the number of inflammatory cells and IL-1β in BALF of mice decrease after CKF treatment. Histopathological observation of lung tissues showed that CKF can remarkably improve alveolar clearance and infiltration of interstitial and alveolar cells after LPS stimulation. In conclusion, our results suggest that CKF inhibits LPS-induced inflammatory response by inhibiting the MyD88/TRIF signaling pathways, thereby protecting mice from LPS-induced ALI.
Acute Lung Injury ; chemically induced ; drug therapy ; genetics ; metabolism ; Adaptor Proteins, Vesicular Transport ; genetics ; metabolism ; Animals ; Anti-Inflammatory Agents ; administration & dosage ; chemistry ; Cardamine ; chemistry ; Cyclooxygenase 2 ; genetics ; metabolism ; Female ; Flowers ; chemistry ; Humans ; Lipopolysaccharides ; adverse effects ; Male ; Mice ; Myeloid Differentiation Factor 88 ; genetics ; metabolism ; NF-kappa B ; genetics ; metabolism ; Nitric Oxide Synthase Type II ; genetics ; metabolism ; Plant Extracts ; administration & dosage ; chemistry ; Signal Transduction ; drug effects ; Tumor Necrosis Factor-alpha ; genetics ; metabolism

OBJECTIVETo investigate the effects of evodiamine (Evo), a component of Evodiaminedia rutaecarpa (Juss.) Benth, on cardiomyocyte hypertrophy induced by angiotensin II (Ang II) and further explore the potential mechanisms.

METHODSCardiomyocytes from neonatal Sprague Dawley rats were isolated and characterized, and then the cadiomyocyte cultures were randomly divided into control, model (Ang II 0.1 μmol/L), and Evo (0.03, 0.3, 3 μmol/L) groups. The cardiomyocyte surface area, protein level, intracellular free calcium ([Ca]) concentration, activity of nitric oxide synthase (NOS) and content of nitric oxide (NO) were measured, respectively. The mRNA expressions of atrial natriuretic factor (ANF), calcineurin (CaN), extracellular signal-regulated kinase-2 (ERK-2), and endothelial nitric oxide synthase (eNOS) of cardiomyocytes were analyzed by real-time reverse transcriptionpolymerase chain reaction. The protein expressions of calcineurin catalytic subunit (CnA) and mitogen-activated protein kinase phosphatase-1 (MKP-1) were detected by Western blot analysis.

RESULTSCompared with the control group, Ang II induced cardiomyocytes hypertrophy, as evidenced by increased cardiomyocyte surface area, protein content, and ANF mRNA expression; increased intracellular free calcium ([Ca]) concentration and expressions of CaN mRNA, CnA protein, and ERK-2 mRNA, but decreased MKP-1 protein expression (P<0.05 or P<0.01). Compared with Ang II, Evo (0.3, 3 μmol/L) significantly attenuated Ang II-induced cardiomyocyte hypertrophy, decreased the [Ca] concentration and expressions of CaN mRNA, CnA protein, and ERK-2 mRNA, but increased MKP-1 protein expression (P<0.05 or P<0.01). Most interestingly, Evo increased the NOS activity and NO production, and upregulated the eNOS mRNA expression (P<0.05).

CONCLUSIONEvo signifificantly attenuated Ang II-induced cardiomyocyte hypertrophy, and this effect was partly due to promotion of NO production, reduction of [Ca]i concentration, and inhibition of CaN and ERK-2 signal transduction pathways.

Angiotensin II ; Animals ; Atrial Natriuretic Factor ; metabolism ; Calcineurin ; genetics ; metabolism ; Calcium ; metabolism ; Dual Specificity Phosphatase 1 ; genetics ; metabolism ; Extracellular Signal-Regulated MAP Kinases ; genetics ; metabolism ; Hypertrophy ; Myocytes, Cardiac ; drug effects ; metabolism ; pathology ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase Type III ; metabolism ; Quinazolines ; pharmacology ; RNA, Messenger ; genetics ; metabolism ; Rats, Sprague-Dawley

Iron deficiency anemia is one of the most common micronutrient deficient conditions around the globe with various consequences, including the weakened immune system. Quercetin is widely distributed bioflavonoid; it has been debated for its dual roles in iron regulation. Quercetin-iron interaction in the body is a complex mechanism which has not been completely understood. The present study aimed to investigate the effect of quercetin on iron supplementation in iron deficiency anemia and on iNOS expression in splenic macrophages. The rat model of iron deficiency anemia was induced by feeding low iron diet to weanling rats for 20 days. The animals were then administered with ferrous sulfate, quercetin, and their combination for 30 days. Blood parameters, histopathological analysis, iron storage, CD68, iNOS and SLC40 expression in rat spleen were investigated. Our results showed that quercetin regulated iron absorption, despite SLC40 down-expression, indicating possible alternate route of iron transport, and that quercetin modulated iNOS production in splenic macrophages.
Anemia, Iron-Deficiency ; drug therapy ; genetics ; metabolism ; Animals ; Dietary Supplements ; analysis ; Female ; Homeostasis ; drug effects ; Humans ; Iron ; deficiency ; Macrophages ; drug effects ; metabolism ; Nitric Oxide Synthase Type II ; genetics ; metabolism ; Quercetin ; administration & dosage ; Rats ; Rats, Sprague-Dawley ; Spleen ; drug effects ; enzymology

Dendrobii Caulis (DC), named 'Shihu' in Chinese, is a precious herb in traditional Chinese medicine. It is widely used to nourish stomach, enhance body fluid production, tonify "Yin" and reduce heat. More than thirty Dendrobium species are used as folk medicine. Some compounds from DC exhibit inhibitory effects on macrophage inflammation. In the present study, we compared the anti-inflammatory effects among eight Dendrobium species. The results provided evidences to support Dendrobium as folk medicine, which exerted its medicinal function partially by its inhibitory effects on inflammation. To investigate the anti-inflammatory effect of Dendrobium species, mouse macrophage cell line RAW264.7 was activated by lipopolysaccharide. The nitric oxide (NO) level was measured using Griess reagent while the pro-inflammatory cytokines were tested by ELISA. The protein expressions of inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2) and mitogen-activated protein kinases (MAPKs) phosphorylation were evaluated by Western blotting analysis. Among the eight Dendrobium species, both water extracts of D. thyrsiflorum B.S.Williams (DTW) and D. chrysotoxum Lindl (DCHW) showed most significant inhibitory effects on NO production in a concentration-dependent manner. DTW also significantly reduced TNF-α, MCP-1, and IL-6 production. Further investigations showed that DTW suppressed iNOS and COX-2 expression as well as ERK and JNK phosphorylation, suggesting that the inhibitory effects of DTW on LPS-induced macrophage inflammation was through the suppression of MAPK pathways. In conclusion, D. thyrsiflorum B.S.Williams was demonstrated to have potential to be used as alternative or adjuvant therapy for inflammation.
Animals ; Anti-Inflammatory Agents ; pharmacology ; Cyclooxygenase 2 ; genetics ; Cytokines ; metabolism ; Dendrobium ; chemistry ; Gene Expression Regulation, Enzymologic ; drug effects ; Inflammation ; chemically induced ; drug therapy ; Lipopolysaccharides ; Macrophages ; drug effects ; enzymology ; Mice ; Mitogen-Activated Protein Kinases ; antagonists & inhibitors ; genetics ; metabolism ; Nitric Oxide ; analysis ; Nitric Oxide Synthase Type II ; genetics ; Phosphorylation ; drug effects ; Plant Extracts ; pharmacology ; RAW 264.7 Cells ; Signal Transduction ; drug effects

Angiotensin II (Ang II) is involved in endothelium injury during the development of hypertension. Tribulus terrestris (TT) is used to treat hypertension, arteriosclerosis, and post-stroke syndrome in China. The present study aimed to determine the effects of aqueous TT extracts on endothelial injury in spontaneously hypertensive rats (SHRs) and its protective effects against Ang II-induced injury in human umbilical vein endothelial cells (HUVECs). SHRs were administered intragastrically with TT (17.2 or 8.6 g·kg·d) for 6 weeks, using valsartan (13.5 mg·kg·d) as positive control. Blood pressure, heart rate, endothelial morphology of the thoracic aorta, serum levels of Ang II, endothelin-1 (ET-1), superoxide dismutase (SOD) and malonaldehyde (MDA) were measured. The endothelial injury of HUVECs was induced by 2 × 10 mol·L Ang II. Cell Apoptosisapoptosis, intracellular reactive oxygen species (ROS) was assessed. Endothelial nitric oxide synthase (eNOS), ET-1, SOD, and MDA in the cell culture supernatant and cell migration were assayed. The expression of hypertension-linked genes and proteins were analyzed. TT decreased systolic pressure, diastolic pressure, mean arterial pressure and heart rate, improved endothelial integrity of thoracic aorta, and decreased serum leptin, Ang II, ET-1, NPY, and Hcy, while increased NO in SHRs. TT suppressed Ang II-induced HUVEC proliferation and apoptosis and prolonged the survival, and increased cell migration. TT regulated the ROS, and decreased mRNA expression of Akt1, JAK2, PI3Kα, Erk2, FAK, and NF-κB p65 and protein expression of Erk2, FAK, and NF-κB p65. In conclusion, TT demonstrated anti-hypertensive and endothelial protective effects by regulating Erk2, FAK and NF-κB p65.
Angiotensin II ; metabolism ; Animals ; Antihypertensive Agents ; administration & dosage ; Apoptosis ; drug effects ; Blood Pressure ; drug effects ; Endothelium, Vascular ; drug effects ; metabolism ; Human Umbilical Vein Endothelial Cells ; drug effects ; Humans ; Hypertension ; drug therapy ; genetics ; metabolism ; physiopathology ; Male ; NF-kappa B ; genetics ; metabolism ; Nitric Oxide Synthase Type III ; genetics ; metabolism ; Oxidative Stress ; drug effects ; Plant Extracts ; administration & dosage ; Proto-Oncogene Proteins c-akt ; genetics ; metabolism ; Rats ; Rats, Inbred SHR ; Rats, Inbred WKY ; Reactive Oxygen Species ; metabolism ; Tribulus ; chemistry

Scutellariae Radix (SR), the root of Scutellaria baicalensis Georgi, is used as an antipyretic drug and has been demonstrated to have anti-inflammatory activity. SR is divided into two specifications, "Ku Qin" (KQ) and "Zi Qin" (ZQ), for use against different symptoms (upper energizer heat or lower portion of the triple energizer), according to the theory of traditional Chinese medicine (TCM). However, differences in the efficacies of these two specifications have not been determined. In the present study, we aimed to characterize the differences in the anti-inflammatory activities between KQ and ZQ and to explore how their differences are manifested in lipopolysaccharide (LPS)-induced macrophages. Our results showed that, in RAW264.7 cells (a mouse macrophage cell line derived from ascites), KQ and ZQ displayed anti-inflammatory effects by inhibiting the release of nitric oxide (NO), inducible NOS (iNOS), and nuclear factor-κB (NF-κB) in a dose-dependent manner without distinction. In NR8383 cells (a rat alveolar macrophage cell line), KQ and ZQ displayed similar effects on NO, iNOS, and NF-κB as seen in RAW264.7 cells, but KQ showed a higher inhibition rate for NO and iNOS than that shown by ZQ at the same concentration. These results indicated that there were differences in efficacy between KQ and ZQ in treating lung inflammation. Our findings provided an experimental evidence supporting the different uses of KQ and ZQ in clinic, as noted in ancient herbal records.
Animals ; Anti-Inflammatory Agents ; pharmacology ; Cell Line ; Lipopolysaccharides ; pharmacology ; Macrophages ; drug effects ; immunology ; Mice ; NF-kappa B ; genetics ; immunology ; Nitric Oxide Synthase Type II ; genetics ; immunology ; RAW 264.7 Cells ; Rats ; Scutellaria baicalensis ; chemistry

BACKGROUNDEndothelial dysfunction is considered as the initiating process and pathological basis of cardiovascular disease. Cyclooxygenase-2 (COX-2) and prostacyclin synthase (PGIS), inducible nitric oxide synthase (iNOS) and endothelial NOS (eNOS) are key enzymes with opposing actions in inflammation and oxidative stress, which are believed to be the major driver of endothelial dysfunction. And in hypoxia (Hx), Hx-inducible factor (HIF)-1α and HIF-2α are predominantly induced to activate vascular endothelial growth factor (VEGF), resulting in abnormal proliferation. Whether and how Tongxinluo (TXL) modulates COX-2, PGIS, iNOS, eNOS, HIF-1α, HIF-2α, and VEGF in Hx-stimulated human cardiac microvascular endothelial cells (HCMECs) have not been clarified.

METHODSHCMEC were treated with CoCl 2 to mimic Hx and the mRNA expressions of COX-2, PGIS, iNOS, eNOS, HIF-1α, HIF-2α, and VEGF were first confirmed, and then their mRNA expression and protein content as well as the cell pathological alterations were evaluated for TXL treatment with different concentrations. In addition, the effector molecular of inflammation prostaglandin E 2 (PGE 2 ) and the oxidative marker nitrotyrosine (NT) was adopted to reflect HCMEC injury.

RESULTSHx could induce time-dependent increase of COX-2, iNOS, HIF-2α, and VEGF in HCMEC. Based on the Hx-induced increase, TXL could mainly decrease COX-2, iNOS, HIF-2α, and VEGF in a concentration-dependent manner, with limited effect on the increase of PGIS and eNOS. Their protein contents verified the mRNA expression changes, which was consistent with the cell morphological alterations. Furthermore, high dose TXL could inhibit the Hx-induced increase of PGE 2 and NT contents, attenuating the inflammatory and oxidative injury.

CONCLUSIONSTXL could inhibit inflammation-related COX-2, oxidative stress-related iNOS, and HIF-2α/VEGF to antagonize Hx-induced HCMEC injury.

Blotting, Western ; Cell Hypoxia ; drug effects ; Cell Line ; Cobalt ; pharmacology ; Drugs, Chinese Herbal ; pharmacology ; Enzyme-Linked Immunosorbent Assay ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; genetics ; metabolism ; Nitric Oxide Synthase Type II ; genetics ; metabolism ; Vascular Endothelial Growth Factor A ; genetics ; metabolism

OBJECTIVEThe study was aimed to investigate the inhibitory effect and mechanism of astragaloside IV (ASI) on the activation of microglial cells.

METHODAfter pre-incubated with ASI for 2 h, microglial cells BV-2 were stimulated with interferon-γ (IFN-γ) for 1. 5 h and 24 h, respectively. Secretion of nitric oxide (NO) in the medium was measured by Griess method. Production of tumor necrosis factor alpha (TNF-α) was detected by ELISA approach. Cellular gene expressions of CD11b, TNF-α, interleukin 1β (IL-1β) and induced nitric oxide synthase (iNOS) were examined by quantitative-PCR analysis. Total and phosphorylation of STAT1, IκB and NF-κB was analyzed by Western blot method.

RESULTASI could significantly inhibit the increased secretion of TNF-α and NO from BV-2 cells upon IFN-γ stimulation (P < 0.001). Further study showed that ASI significantly down-regulated gene expression of IL-1β and TNF-α (P < 0.01, P < 0.05) and exhibited a trend to reduce that of iNOS. IFN-γ and ASI have no obvious effect on gene expression of CD11b. Moreover, ASI inhibited the phosphorylation of STAT1, IκB and NF-κB elicited by IFN-γ stimulation.

CONCLUSIONASI could restrain microglial activation through interfering STAT1/IκB/NF-κB signaling pathway, reducing gene expres- sion of IL-1β and TNF-α, and thus inhibiting the production of proinflammatory mediators such as NO and TNF-α.

Animals ; Astragalus Plant ; chemistry ; Drugs, Chinese Herbal ; pharmacology ; I-kappa B Proteins ; genetics ; metabolism ; Interferon-gamma ; genetics ; metabolism ; Mice ; NF-kappa B ; genetics ; metabolism ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase Type II ; genetics ; metabolism ; STAT1 Transcription Factor ; genetics ; metabolism ; Saponins ; pharmacology ; Signal Transduction ; drug effects ; Triterpenes ; pharmacology

OBJECTIVETo explore the diaphragmatic toxicity in doxorubicin (DOX)-treated rats and the related mechanisms, as well as the effects of Shengmai Injection (SMI, ) on the diaphragmatic dysfunction.

METHODSThirty Sprague-Dawley male rats were randomly divided into three groups: control, DOX-treated and DOX+SMI treated groups. DOX was given to rats in DOX and DOX+SMI groups in 6 equal doses [2.5 mg/kg, intraperitoneal injection (i.p.)], on alternate days, over a period of 2 weeks for a cumulative dose of 15 mg/kg. SMI was given to DOX+SMI rats in 12 doses (3 mL/kg, i.p.) for a period of 2 weeks before the administration of DOX and 2 weeks during the administration of DOX. The rats in the control group received equal volume of normal saline. Subsequently, the twitch and tetanic characteristics and force-frequency relationships, and the malondialdehyde (MDA) levels and the superoxide dismutase (SOD) activities, as well as the mRNA content and proteins of inducible nitric oxide synthase (iNOS) were determined.

RESULTSThe DOX-treated rats had decreased the peak twitch tension (Pt), maximal tetanic tension (P0) and force-frequency relationship as compared with the control rats (P<0.01), while the diaphragm contractility in rats treated with SMI were significantly higher than that in DOX-treated rats (P<0.01). The DOX-treated rats had increased MAD levels and decreased SOD activities (P<0.05), and SMI decreased the MDA levels and increased the SOD activities in DOX-treated rats (P<0.05). Ultrastructure of diaphragm in the DOX-treated rats revealed typical alterations including fracture of diaphragm fibers, and edema and degeneration of mitochondria; these changes were relieved by SMI treatment. The mRNA content and protein of iNOS in DOX-treated rats were remarkably higher than those in control rats (P<0.01), while SMI decreased the mRNA expression level of iNOS in DOX-treated rats (P<0.05).

CONCLUSIONSLipid peroxidation is responsible for DOX-induced diaphragm toxicity. SMI protects diaphragm muscles and their function from DOX impairment, and these beneficial effects may be somehow correlated with the decrease in expression of iNOS and lipid peroxidation.

Animals ; Biomechanical Phenomena ; drug effects ; Blotting, Western ; Diaphragm ; drug effects ; pathology ; physiology ; ultrastructure ; Doxorubicin ; adverse effects ; Drugs, Chinese Herbal ; pharmacology ; Gene Expression Regulation ; drug effects ; In Vitro Techniques ; Injections ; Male ; Malondialdehyde ; metabolism ; Muscle Contraction ; drug effects ; Nitric Oxide Synthase Type II ; genetics ; metabolism ; Oxidative Stress ; drug effects ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo evaluate the protective effects of Reduning Injection (, RDN), a patent Chinese medicine, on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in rats and its underlying mechanisms of action.

METHODSSixty male Sprague-Dawley rats were randomly divided into 6 groups, including normal control, model, dexamethasone (DEX, 5 mg/kg), RDN-H (720 mg/kg), RDN-M (360 mg/kg) and RDN-L (180 mg/kg) groups, with 10 rats in each group. Rats were challenged with intravenous injection of LPS 1 h after intraperitoneal treatment with RDN or DEX. At 6 h after LPS challenge, lung tissues and bronchoalveolar lavage fluid (BALF) were collected, and the number of inflammatory cells was determined. The right lungs were collected for histopathologic examination, measurement of gene and protein expressions, superoxide dismutase (SOD) and myeloperoxidase (MPO) activities.

RESULTSIn vivo pretreatment of RDN (360, 720 mg/kg) significantly reduced the weight of wet to dry (W/D) ratio of lung, protein content in BALF, and led to remarkable attenuation of LPS-induced histopathological changes in the lungs. Meanwhile, RDN enormously decreased BALF total inflammatory cells, especially neutrophil and macrophage cell numbers. Moreover, RDN increased SOD activity, inhibited MPO activity, alleviated LPS-induced tumor neurosis factor-α (TNF-α) and inducible nitric oxide synthase (iNOS) expression in lung tissues. Furthermore, RDN (720 mg/kg) efficiently weakened nuclear factorkappa B (NF-κB) gene and protein expression.

CONCLUSIONAnti-inflammatory effects of RDN was demonstrated to be preventing pulmonary neutrophil infiltration, lowering MPO activity, TNF-α and iNOS gene expression by inhibiting NF-κB activity in LPS-induced ALI.

Acute Lung Injury ; drug therapy ; enzymology ; pathology ; Animals ; Anti-Inflammatory Agents ; administration & dosage ; chemistry ; pharmacology ; therapeutic use ; Bronchoalveolar Lavage Fluid ; cytology ; Cell Count ; Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal ; administration & dosage ; chemistry ; pharmacology ; therapeutic use ; Gene Expression Regulation ; drug effects ; Injections ; Lipopolysaccharides ; Lung ; drug effects ; enzymology ; pathology ; Male ; NF-kappa B ; genetics ; metabolism ; Nitric Oxide Synthase Type II ; genetics ; metabolism ; Peroxidase ; metabolism ; Rats, Sprague-Dawley ; Superoxide Dismutase ; metabolism ; Tumor Necrosis Factor-alpha ; genetics ; metabolism