Extract of Pinus densiflora needles suppresses acute inflammation by regulating inflammatory mediators in RAW264.7 macrophages and mice

ContextPinus densiflora Siebold & Zucc. (Pinaceae) needle extracts ameliorate oxidative stress, but research into their anti-inflammatory effects is limited.ObjectiveTo investigate antioxidant and anti-inflammatory effects of a Pinus densiflora needles (PINE) ethanol extract in vitro and in vivo.Materials and methodsWe measured levels of reactive oxygen species (ROS), superoxide dismutase (SOD) and inflammatory mediators in lipopolysaccharide (LPS)-stimulated RAW264.7 cells at various PINE concentrations (25, 50 and 100 μg/mL; but 6.25, 12.5 and 25 μg/mL for interleukin-1β and prostaglandin E₂ (PGE₂)). Thirty ICR mice were randomized to six groups: vehicle, control, PINE pre-treatment (0.1, 0.3 and 1 mg/left ear for 10 min followed by arachidonic acid treatment for 30 min) and dexamethasone. The posttreatment ear thickness and myeloperoxidase (MPO) activity were measured.ResultsPINE 100 μg/mL significantly decreased ROS (IC₅₀, 70.93 μg/mL, p < 0.01), SOD (IC₅₀, 30.99 μg/mL, p < 0.05), malondialdehyde (p < 0.01), nitric oxide (NO) (IC₅₀, 27.44 μg/mL, p < 0.01) and tumour necrosis factor-alpha (p < 0.05) levels. Interleukin-1β (p < 0.05) and PGE₂ (p < 0.01) release decreased significantly with 25 μg/mL PINE. PINE 1 mg/ear inhibited LPS-stimulated expression of cyclooxygenase-2 and inducible NO synthase in RAW264.7 macrophages and significantly inhibited ear oedema (36.73–15.04% compared to the control, p < 0.01) and MPO activity (167.94–105.59%, p < 0.05).Discussion and conclusionsPINE exerts antioxidant and anti-inflammatory effects by inhibiting the production of inflammatory mediators. Identified flavonoids such as taxifolin and quercetin glucoside can be attributed to effect of PINE.     

Anti-Inflammatory Effect of Mangostenone F in Lipopolysaccharide-Stimulated RAW264.7 Macrophages by Suppressing NF-κB and MAPK Activation

Mangostenone F (MF) is a natural xanthone isolated from Garcinia mangostana. However, little is known about the biological activities of MF. This study was designed to investigate the anti-inflammatory effect and underlying molecular mechanisms of MF in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. MF dose-dependently inhibited the production of NO, iNOS, and pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β) in LPS-stimulated RAW264.7 macrophages. Moreover, MF decreased the NF-κB luciferase activity and NF-κB DNA binding capacity in LPS-stimulated RAW264.7 macrophages. Furthermore, MF suppressed the NF-κB activation by inhibiting the degradation of IκBα and nuclear translocation of p65 subunit of NF-κB. In addition, MF attenuated the AP-1 luciferase activity and phosphorylation of ERK, JNK, and p38 MAP kinases. Taken together, these results suggest that the anti-inflammatory effect of MF is associated with the suppression of NO production and iNOS expression through the down-regulation of NF-κB activation and MAPK signaling pathway in LPS-stimulated RAW264.7 macrophages.     

β-Hydroxyisovalerylshikonin regulates macrophage polarization via the AMPK/Nrf2 pathway and ameliorates sepsis in mice

ContextThe potential anti-inflammatory bioactivities of β-hydroxyisovalerylshikonin (β-HIVS) remain largely unknown.ObjectiveThis study investigated the anti-inflammatory effects and underlying mechanisms of β-HIVS.Materials and methodsRAW 264.7 cells stimulated with LPS (100 ng/mL) for 24 h were treated with the non-cytotoxic doses of β-HIVS (0.5 or 1 μM, determined by MTT and Trypan blue staining), qRT-PCR and FCM assay were used to examine macrophage polarization transitions. Western blotting was used to evaluate the activation of the AMPK/Nrf2 pathway. In vivo, C57BL/6 mice were randomly divided into vehicle control, LPS (10 mg/kg), and β-HIVS (2.5 mg/kg) combined with LPS (10 mg/kg) groups, blood samples, BALF, and lung tissues of mice were subjected to ELISA, qRT-PCR, FCM, and H&E staining.Resultsβ-HIVS (1 μM) inhibited LPS-induced expression of M1 macrophage markers (TNF-α: 0.29-fold, IL-1β: 0.32-fold), promoted the expression of M2 macrophage markers (CD206: 3.14-fold, Arginase-1: 3.98-fold) in RAW 264.7 cells; mechanistic studies showed that β-HIVS increased the expression of nuclear Nrf2 (2.04-fold) and p-AMPK (3.65-fold) compared with LPS group (p < 0.05). In vivo, β-HIVS decreased the levels of pro-inflammatory cytokines (TNF-α: 1130.41 vs. 334.88 pg/mL, IL-1β: 601.89 vs. 258.21 pg/mL in serum; TNF-α: 893.07 vs. 418.21 pg/mL, IL-1β: 475.22 vs. 298.54 pg/mL in BALF), decreased the proportion of M1 macrophages (77.83 vs. 68.53%) and increased the proportion of M2 macrophages (13.55 vs. 19.56%) in BALF, and reduced lung tissue damage and septic mice survival (p < 0.05).ConclusionsThese results indicate that β-HIVS may be a new potential anti-inflammatory agent.     

Silk fibroin hydrogel containing Sesbania sesban L. extract for rheumatoid arthritis treatment

PurposeRheumatoid arthritis, a chronic and progressive inflammation condition in the joints, has significantly reduced the patient quality of life and life expectancy. Crucially, there is no complete therapy for this disease, and the current treatments possess numerous side effects. Thus, novel therapeutic approach is necessary. To that end, this study developed novel silk fibroin in-situ hydrogel containing Sesbania sesban L. extract, a plant with high anti-inflammatory actions that are beneficial for rheumatoid arthritis treatments.MethodsThe hydrogels were manufactured using simple method of spontaneous gelation at different temperature. The gel properties of morphology, gelation time, viscosity, gel strength, stability, drug loading capacity, drug release rate, and in-vitro anti-inflammatory activity were investigated with appropriate methods.ResultsThe optimal formulation had highly porous structure, with a gelation time of 0.5 h at room temperature and bodily temperature of 37 °C, a viscosity of 2530 ± 50 cP, a gel strength of 1880.14 ± 35.10 g, and a physical stability of >6 months. Moreover, the hydrogel contained the Sesbania sesban L. leaf extract with a total phenolic content of 92.8 ± 8.30 mg GAE/g, and sustained the release rate for >20 dạys, followed the Higuchi model. Regarding the in-vitro activities, all formulations were nontoxic to the RAW 264.7 cell line and demonstrated comparable anti-inflammatory activity to the free extract, in terms of the NO reduction levels.ConclusionConclusively, the systems possessed potential properties to be further investigated to become a prospective rheumatoid arthritis treatment.     

Polymeric versus lipid nanocapsules for miconazole nitrate enhanced topical delivery: in vitro and ex vivo evaluation

Nanocapsules can be equated to other nanovesicular systems in which a drug is entrapped in a void containing liquid core surrounded by a coat. The objective of the present study was to investigate the potential of polymeric and lipid nanocapsules (LNCs) as innovative carrier systems for miconazole nitrate (MN) topical delivery. Polymeric nanocapsules and LNCs were prepared using emulsification/nanoprecipitation technique where the effect of poly(ε-caprolactone (PCL) and lipid matrix concentrations with respect to MN were assessed. The resulted nanocapsules were examined for their average particle size, zeta potential, %EE, and in vitro drug release. Optimum formulation in both polymeric and lipidic nanocapsules was further subjected to anti-fungal activity and ex vivo permeation tests. Based on the previous results, nanoencapsulation strategy into polymeric and LNCs created formulations of MN with slow biphasic release, high %EE, and improved stability, representing a good approach for the delivery of MN. PNCs were best fitted to Higuchi’s diffusion while LNCs followed Baker and Lonsdale model in release kinetics. The encapsulated MN either in PNCs or LNCs showed higher cell viability in WISH amniotic cells in comparison with free MN. PNCs showed less ex vivo permeation. PNCs were accompanied by high stability and more amount drug deposition (32.2 ± 3.52 µg/cm²) than LNCs (12.7 ± 1.52 µg/cm²). The antifungal activity of the PNCs was high 19.07 mm compared to 11.4 mm for LNCs. In conclusion, PNCs may have an advantage over LNCs by offering dual action for both superficial and deep fungal infections.     

Oridonin ameliorates inflammation-induced bone loss in mice via suppressing DC-STAMP expression

Currently, dendritic cell-specific transmembrane protein (DC-STAMP), a multipass transmembrane protein, is considered as the master regulator of cell–cell fusion, which underlies the formation of functional multinucleated osteoclasts. Thus, DC-STAMP has become a promising target for osteoclast-associated osteolytic diseases. In this study, we investigated the effects of oridonin (ORI), a natural tetracyclic diterpenoid compound isolated from the traditional Chinese herb Rabdosia  rubescens, on osteoclastogenesis in vivo and ex vivo. ICR mice were injected with LPS (5 mg/kg, ip, on day 0 and day 4) to induce inflammatory bone destruction. Administration of ORI (2, 10 mg·kg⁻¹·d⁻¹, ig, for 8 days) dose dependently ameliorated inflammatory bone destruction and dramatically decreased DC-STAMP protein expression in BMMs isolated from LPS-treated mice. Treatment of preosteoclast RAW264.7 cells with ORI (0.78–3.125 μM) dose dependently inhibited both mRNA and protein levels of DC-STAMP, and suppressed the following activation of NFATc1 during osteoclastogenesis. Knockdown of DC-STAMP in RAW264.7 cells abolished the inhibitory effects of ORI on RANKL-induced NFATc1 activity and osteoclast formation. In conclusion, we show for the first time that ORI effectively attenuates inflammation-induced bone loss by suppressing DC-STAMP expression, suggesting that ORI is a potential agent against inflammatory bone diseases.     

3‑Bromo‑5‑(ethoxymethyl)‑1,2‑benzenediol inhibits LPS-induced pro-inflammatory responses by preventing ROS production and downregulating NF-κB in vitro and in a zebrafish model

The anti-inflammatory effects of 3‑bromo‑5‑(ethoxymethyl)‑1,2‑benzenediol (BEMB) from Polysiphonia morrowii were evaluated in lipopolysaccharide (LPS)-induced RAW264.7 cells and zebrafish embryo. BEMB showed anti-inflammatory effects by inhibiting the production of nitric oxide (NO) and reactive oxygen species (ROS), and the expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) in the LPS-activated RAW264.7 cells and zebrafish embryo without cytotoxicity. Moreover, BEMB suppressed the protein and mRNA expression levels of nuclear factor (NF)-κB (p65 and inhibitor of NF-κB [IκB]-A) in RAW264.7 cells and zebrafish embryo, respectively. Collectively, the results of this study indicate that BEMB suppressed the production of pro-inflammatory mediators such as NO, iNOS, and COX-2 as well as their regulation in LPS-induced RAW264.7 cells and zebrafish embryos by inhibiting ROS production and NF-κB expression. Therefore, this study suggests that BEMB could be a potential anti-inflammatory agent for the treatment of inflammatory diseases.     

FAEE exerts a protective effect against osteoporosis by regulating the MAPK signalling pathway

ContextFerulic acid ethyl ester (FAEE) is abundant in Ligusticum chuanxiong Hort. (Apiaceae) and grains, and possesses diverse biological activities; but the effects of FAEE on osteoporosis has not been reported.ObjectiveThis study investigated whether FAEE can attenuate osteoclastogenesis and relieve ovariectomy-induced osteoporosis via attenuating mitogen-activated protein kinase (MAPK).Materials and methodsWe stimulated RAW 264.7 cells with receptor activator of NF-κB ligand (RANKL) followed by FAEE. The roles of FAEE in osteoclast production and osteogenic resorption of mature osteoclasts were evaluated by tartrate resistant acid phosphatase (TRAP) staining, expression of osteoclast-specific genes, proteins and MAPK. Ovariectomized (OVX) female Sprague-Dawley rats were administered FAEE (20 mg/kg/day) for 12 weeks to explore its potential in vivo, and then histology was undertaken in combination with cytokines analyses.ResultsFAEE suppressed RANKL-induced osteoclast formation (96 ± 0.88 vs. 15 ± 1.68) by suppressing the expression of osteoclast-specific genes, proteins and MAPK signalling pathway related proteins (p-ERK/ERK, p-JNK/JNK and p-P38/P38) in vitro. In addition, OVX rats exposed to FAEE maintained their normal calcium (Ca) (2.72 ± 0.02 vs. 2.63 ± 0.03, p < 0.05) balance, increased oestradiol levels (498.3 ± 9.43 vs. 398.7 ± 22.06, p < 0.05), simultaneously reduced levels of bone mineral density (BMD) (0.159 ± 0.0016 vs. 0.153 ± 0.0025, p < 0.05) and bone mineral content (BMC) (0.8 ± 0.0158 vs. 0.68 ± 0.0291, p < 0.01).Discussion and conclusionsThese findings suggested that FAEE could be used to ameliorate osteoporosis by the MAPK signalling pathway, suggesting that FAEE could be a potential therapeutic candidate for osteoporosis.     

Anti-Inflammatory and Anti-Superbacterial Properties of Sulforaphane from Shepherd's Purse

Shepherd''s purse, Capsella bursa-pastoris (L.) Medik., has been considered a health food for centuries in Asia and is known to contain the isothiocyanate compound sulforaphane. In this study, we evaluated the anti-inflammatory and antibacterial properties of a sulforaphane-containing solution (SCS) isolated from shepherd''s purse. SCS had significant anti-inflammatory activity indicated by the decreased levels of nitric oxide (NO), cytokines (interleukin 1β [IL-1β], IL-6, and IL-10), and prostaglandin E₂ (PGE₂) in lipopolysaccharide-stimulated RAW 264.7 murine macrophages. In addition, SCS decreased the inducible NO synthase (iNOS) and cyclooxygenase 2 (COX-2) levels, which confirmed the anti-inflammatory activity of SCS. Further, SCS inhibited vancomycin-resistant enterococci (VRE) and Bacillus anthracis. The minimal inhibitory concentration was 250 µg/ml for VRE and 1,000 µg/ml for B. anthracis. Taken together, these data indicate that SCS has potential anti-inflammatory and anti-superbacterial properties, and thus it can be used as a functional food or pharmaceutical.     

Sauropus brevipes ethanol extract negatively regulates inflammatory responses in vivo and in vitro by targeting Src,Syk and IRAK1

ContextSauropus brevipes Müll. Arg. (Phyllanthaceae) has been used as an effective ingredient in a decoction for the treatment of diarrhoea. However, there was no report on its modulatory role in inflammation.ObjectiveThis study investigates anti-inflammatory effect of S. brevipes in various inflammation models.Materials and methodsThe aerial part of S. brevipes was extracted with 95% ethanol to produce Sb-EE. RAW264.7 cells pre-treated with Sb-EE were stimulated by lipopolysaccharide (LPS), and Griess assay and PCR were performed. High-performance liquid chromatography (HPLC) analysis, luciferase assay, Western blotting and kinase assay were employed. C57BL/6 mice (10 mice/group) were orally administered with Sb-EE (200 mg/kg) once a day for five days, and peritonitis was induced by an intraperitoneal injection of LPS (10 mg/kg). ICR mice (four mice/group) were orally administered with Sb-EE (20 or 200 mg/kg) or ranitidine (positive control) twice a day for two days, and EtOH/HCl was orally injected to induce gastritis.ResultsSb-EE suppressed nitric oxide (NO) release (IC₅₀=34 µg/mL) without cytotoxicity and contained flavonoids (quercetin, luteolin and kaempferol). Sb-EE (200 µg/mL) reduced the mRNA expression of inducible NO synthase (iNOS). Sb-EE blocked the activities of Syk and Src, while inhibiting interleukin-1 receptor associated kinases (IRAK1) by 68%. Similarly, orally administered Sb-EE (200 mg/kg) suppressed NO production by 78% and phosphorylation of Src and Syk in peritonitis mice. Sb-EE also decreased inflammatory lesions in gastritis mice.Discussion and conclusionsThis study demonstrates the inhibitory effect of Sb-EE on the inflammatory response, suggesting that Sb-EE can be developed as a potential anti-inflammatory agent.     

Aqueous core epigallocatechin gallate PLGA nanocapsules: characterization,antibacterial activity against uropathogens,and in vivo reno-protective effect in cisplatin induced nephrotoxicity

Epigallocatechin-3-gallate (EGCG) was isolated from Cycas thouarsii leaves for the first time and encapsulated in aqueous core poly(lactide-co-glycolide) (PLGA) nanocapsules (NCs). This work investigates antimicrobial activity and in vivo reno-protective effects of EGCG-PLGA NCs in cisplatin-induced nephrotoxicity. A double emulsion solvent evaporation process was adopted to prepare PLGA NCs loaded with EGCG. Particle size, polydispersity index (PDI), zeta potential, percent entrapment efficiency (%EE), structural morphology, and in vitro release platform were all studied in vitro. The optimum formula (F2) with particle size (61.37 ± 5.90 nm), PDI (0.125 ± 0.027), zeta potential (–11.83 ± 3.22 mV), %EE (85.79 ± 5.89%w/w), initial burst (36.85 ± 4.79), and percent cumulative release (87.79 ± 9.84) was selected for further in vitro/in vivo studies. F2 exhibited an enhanced antimicrobial activity against uropathogens as it had lower minimum inhibitory concentration (MIC) values and a more significant impact on bacterial growth than free EGCG. Forty male adult mice were randomly allocated into five groups: control vehicle, untreated methotrexate, MTX groups treated with a daily oral dose of free EGCG, placebo PLGA NCs, and EGCG PLGA NCs (F2) for 10 days. Results showed that EGCG PLGA NCs (F2) exerted promising renoprotective effects compared to free EGCG. EGCG PLGA NCs group induced a significant decrease in kidney index, serum creatinine, kidney injury molecule-1 (KIM-1), NGAL serum levels, and pronounced inhibition of NLPR-3/caspase-1/IL/1β inflammasome pathway. It also significantly ameliorated oxidative stress and decreased NFκB, Bax expression levels. Aqueous core PLGA NCs are a promising formulation strategy that provides high polymeric protection and sustained release pattern for hydrophilic therapeutic agents.     

NCX 2057, a novel NO-releasing derivative of ferulic acid,suppresses inflammatory and nociceptive responses in in vitro and in vivo models

Background and purpose:

We previously reported that NCX 2057, a compound comprising a nitric oxide (NO)-releasing moiety and the natural antioxidant, ferulic acid (FA), inhibits pro-inflammatory mediators through NO-mediated gene regulation. Here, we have assessed the activities of NCX 2057 in models of inflammatory and neuropathic pain, and characterized its effects on cyclooxygenase (COX)-1 and COX-2.

Experimental approach:

Anti-nociceptive and anti-inflammatory activities of NCX 2057 were measured in vitro and in vivo in models of inflammatory (carrageenan) and neuropathic (chronic constriction injury; CCI) pain. Effects of NCX 2057 were measured on COX-1 and COX-2 activities in RAW 264.7 macrophages.

Key results:

NCX 2057 dose-dependently inhibited single motor unit responses to noxious mechanical stimulation (ID₅₀= 100 µmol·kg⁻¹) and wind-up responses in rats with paw inflammation induced by carrageenan. Moreover, NCX 2057 inhibited allodynic responses following CCI of the sciatic nerve [ipsilateral Paw Withdrawal Threshold (g): vehicle: 41.4 ± 3.3; NCX 2057: 76.3 ± 4.8 FA: 37.9 ± 15.5 at 175 µmol·kg⁻¹]. NCX 2057 reversed carrageenan-induced hyperalgesic responses in mice and inhibited prostaglandin E₂ formation in paw exudates. Finally, NCX 2057 competitively inhibited COX-1 and COX-2 activities in whole RAW macophages (IC₅₀= 14.7 ± 7.4 and 21.6 ± 7.5 µM, respectively). None of these properties were exhibited by equivalent treatments with FA or standard NO donor compounds.

Conclusions and implications:

These studies indicate that NCX 2057 is effective in chronic inflammatory and neuropathic pain models, probably because of its particular combination of anti-COX, antioxidant and NO-releasing properties.     

Identification of sterols from the soft coral Dendronephthya gigantea and their anti-inflammatory potential

Exploration of anti-inflammatory phytochemicals has received tremendous attention worldwide owing to the rapid increase in inflammatory diseases. Current study reveals the identification of eight 3β-hydroxy-Δ⁵-steroidal congeners from a nonpolar column fraction of the ethanol solubles from the soft coral Dendronephthya gigantea collected from Jeju Island South Korea, using GC–MS/MS analysis. The sterol-rich fraction (DGEH21) showed a significant anti-inflammatory activity as exhibited by the inhibition of NO production (IC₅₀ 4.33 ± 0.50 μg/mL) and PGE₂ production in LPS-stimulated RAW 264.7 macrophages. It also suppressed the expression levels of proinflammatory cytokines, TNF-α, IL-1β, and IL-6 in a dose-dependent manner. Furthermore, DGEH21 effectively downregulated the expression levels of iNOS, and COX-2 and reduced NO and ROS production as well as cell death in LPS-stimulated in-vivo zebrafish embryo model. However, DGEH21 at relatively high concentrations indicated cytotoxicity in both RAW cells and zebrafish embryos with RAW cell viability being nearly 80% after treatment with 25 μg/mL DGEH21. This study highlights the synergistic anti-inflammatory activity of several steroids found in D. gigantea. Their actions may be useful in the development of anti-inflammatory cosmeceuticals, pharmaceutical agents, and other consumer products.     

Chemical composition and anti-inflammatory activities of essential oil from Trachydium roylei

Chemical composition, anti-inflammatory activity, and cytotoxicity of essential oils obtained from the aerial parts of Trachydium roylei were investigated in this study. The chemical composition of T. roylei essential oil was analyzed using gas chromatography mass spectrometry. Fifty-nine components, representing 98.87% of the oils, were characterized. The oils were predominated by aromatic compounds and monoterpene hydrocarbons, and the main components were myristicin (25.35%), β-phellandrene (22.95%), elemicine (7.69%), isoelemicin (5.48%), and cedrol (5.26%). The anti-inflammatory activity of the oil in lipopolysaccharide-stimulated murine RAW 264.7 cells was evaluated. The oils downregulated the production of proinflammatory cytokines, including tumor necrosis factor-α, interleukin (IL)-1β, and IL-6, and significantly increased the anti-inflammatory cytokine IL-10 levels. Results indicated that the oils effectively inhibited the secretion of nitric oxide and prostaglandin E2 in lipopolysaccharide-stimulated macrophages. Western blot analyses were performed to determine whether the inhibitory effects of the oils on proinflammatory mediators (nitric oxide and prostaglandin E2) were related to the modulation of inducible nitric oxide synthase and cyclooxygenase-2 expression. These findings suggest that T. roylei essential oils exert an anti-inflammatory effect by regulating the expression of inflammatory cytokines.     

白木香叶提取物对LPS诱导RAW264.7巨噬细胞炎症因子的影响

目的 采用LPS刺激巨噬细胞RAW264.7,建立体外的炎症模型,探讨白木香叶提取物抗炎活性和作用机制.方法 采用MTT法检测白木香叶提取物( ASPE)对RAW264.7细胞的毒性作用;采用ELISA法检测ASPE对IL-6表达情况;采用Western blotting检测iNOS和COX-2蛋白表达情况,结果 ASPE能抑制LPS刺激RAW264.7所产生的炎症反应,其作用机制可能与其抑制iNOS和COX-2蛋白表达有关.结论 白木香叶提取物有抗炎活性.     

Andrographolide suppresses the expression of inducible nitric oxide synthase in macrophage and restores the vasoconstriction in rat aorta treated with lipopolysaccharide

1. We investigated whether andrographolide, a diterpenoid lactone found at Andrographis paniculata, influences the induction of the inducible nitric oxide synthase (iNOS) in RAW264.7 cells activated by bacterial endotoxin (LPS), as well as in the rats with endotoxic shock and in aortic rings treated with LPS.
2. Incubation of RAW264.7 cells with andrographolide (1 to 50 μM) inhibited the LPS (1 μg ml⁻¹)-induced nitrite accumulation in concentration- and time-dependent manners. Maximum inhibition was observed when andrographolide was added together with LPS and decreased progressively as the interval between andrographolide and LPS was increased to 20 h.
3. Western blot analysis demonstrated that iNOS expression was markedly attenuated in the presence of andrographolide for 6–24 h, suggesting that andrographolide inhibited iNOS protein induction.
4. Thoracic aorta incubation with LPS (300 ng ml⁻¹) for 5 h in vitro exhibited a significant decrease in the maximal contractile response to phenylephrine (10⁻⁹–10⁻⁵ M). Andrographolide (30 μM) restored the contractile response to control level.
5. In anaesthetized rats, LPS (10 mg kg⁻¹, i.v.) caused a fall in mean arterial blood pressure (MAP) from 116±4 to 77±5 mmHg. The pressor effect of phenylephrine (10 μg ml⁻¹, i.v.) was also significantly reduced at 30, 60, 120 and 180 min after LPS injection. In contrast, animals pretreated with andrographolide (1 mg kg⁻¹, i.v., 20 min prior to LPS) maintained a significantly higher MAP when compared to LPS-rats given with vehicle. Administration of andrographolide 60 min after LPS caused a increase in MAP and significantly reversed the reduction of the pressor response to phenylephrine.
6. Our results indicated that andrographolide inhibits nitrite synthesis by suppressing expression of iNOS protein in vitro. And, this inhibition of iNOS synthesis may contribute to the beneficial haemodynamic effects of andrographolide in endotoxic shock.

     

Yiguanjian decoction inhibits macrophage M1 polarization and attenuates hepatic fibrosis induced by CCl4/2-AAF

ContextOur previous studies indicated that Yiguanjian decoction (YGJ) has an anti-hepatic-fibrosis effect and could regulate macrophage status.ObjectiveTo elucidate the mechanism of YGJ in regulating macrophages.Materials and methodsLiver cirrhosis was induced by CCl₄ for 12 weeks combined with 2-acetylaminofluorene (2-AAF) for the last 4 weeks in male Wistar rats. YGJ (3.56 mg/kg) orally administered in the last 4 weeks, and SORA (1 mg/kg) as control. In vitro, RAW264.7 cells were treated with lipopolysaccharides (LPSs) to induce macrophage polarization to the M1 phenotype, and they were co-cultured with WB-F344 cells and allocated to M group, YGJ group (2 μg/mL) and WIF-1 group (1 μg/mL) with untreated cells as control. The differentiation direction of WB-F344 cell line was observed in the presence or absence of YGJ. Pathology, fibrosis-related cytokines, macrophage polarization-related components, and Wnt signalling pathway components were detected.ResultsIn vivo, the expression levels of α-SMA, Col (1), OV6, SOX9, EpCAM and M1 macrophage-related components (STAT1, IRF3, IRF5, IRF8, SOCS3) significantly decreased in the YGJ group compared with those in the 2-AAF/CCl₄ group (p < 0.01 or 0.05). In vitro, the expression levels of M1 macrophage-related components, including STAT1, NF-κB, IRF3, IRF5, and SOCS3, in RAW264.7 cells decreased significantly in the YGJ group compared with those in the M group (p < 0.05 or p < 0.01). The expression levels of Wnt3A, FZD5, LRP-5/-6, and β-catenin significantly increased in the YGJ group compared with those in the M group (p < 0.05 or p < 0.01). In addition, the expression levels of Wnt-4/-5A/-5B, and FZD2 significantly decreased in the YGJ group compared with those in the M group (p < 0.05 or p < 0.01).ConclusionThis study suggests that the anti-cirrhosis effect of YGJ is associated with its ability to inhibit macrophage M1-polarization, which provides a scientific basis for the clinical application of YGJ.