Dehydroandrographolide targets CD300f and negatively regulated MRGPRX2-induced pseudo-allergic reaction.

Mas-related G protein-coupled receptor X2 (MRGPRX2) mediates mast cells (MCs) activation, which is a key target for the treatment of allergic diseases. However, there are few drugs targeting MRGPRX2. Leukocyte mono-immunoglobulin-like receptor 3 (CD300f) is a negative regulator of FcεRΙ-mediated MC activation. However, the regulatory effect of CD300f on MRGPRX2 remains unclear. Dehydroandrographolide (DA) is a main contributor of Andrographis paniculata (Burm.f.) Nees (family: Acanthaceae) have been shown to inhibit type I hypersensitivity. The aim of this study was to determine whether DA negatively regulated MRGPRX2-mediated MC activation via CD300f and showed therapeutic effect on pseudo-allergic reactions. Mouse allergic models and MC degranulation were detected in vivo and in vitro, and inflammatory mediators were detected. siRNA interference and Biacore were used to verify the target. DA inhibited pseudo-allergic reactions by reducing vasodilation and serum cytokine levels in mice and inhibited MRGPRX2-mediated MC activation. The regulatory effect of DA was significantly decreased after the knockdown of CD300f expression. Moreover, DA upregulated the phosphorylation level of Src homology region 2 domain-containing phosphatase (SHP)-1 and SHP-2, which are key kinases in the negative regulatory signaling pathways associated with CD300f. In conclusion, DA negatively regulates MRGPRX2-mediated MC activation via CD300f to inhibit pseudo-allergic reactions.

Dehydroandrographolide inhibits IgE-mediated anaphylactic reactions via calcium signaling pathway.

The classical mast cells degranulation pathway is mediated by FcεRI aggregation and varies in strength among subjects. Dehydroandrographolide (DA) is one of principal components of Andrographis paniculata (Burm.f.) Nees (family: Acanthaceae) and considered the main contributors of its therapeutic properties, such as anti-tumor. In this study, inhibition of IgE-mediated anaphylactic reactions and anti-inflammatory potential of DA were investigated. The anti-anaphylactic activity of DA was investigated using skin swelling and extravasation assays in vivo and mast cell degranulation assay in vitro. The release of cytokines was measured using ELISA kits. Human Phospho-Kinase Array kit and western blotting were used to explore the related molecular signaling pathways. DA inhibited IgE-mediated mast cell activation, including degranulation and release of cytokines in vitro. Moreover, DA reduced the degree of swelling and Evans blue exudation of mice paw in a dose-dependent manner by inhibiting mast cell degranulation. DA obviously reduced the concentrations of histamine, TNF-α, MCP-1, IL-8, IL-13, and IL-4 in mice serum and inhibited IgE-mediated anaphylactic reactions as a potential P-PLCγ inhibitor. Our study reveals that DA can inhibit allergic responses in vivo and in vitro, and it may be regarded as a novel P-PLCγ inhibitor for preventing mast cell-immediate and delayed allergic diseases.

Anti-Inflammatory Activity of Dehydroandrographolide by TLR4/NF-κB Signaling Pathway Inhibition in Bile Duct-Ligated Mice.

BACKGROUND/AIMS: Clinically, biliary obstruction is often accompanied by progressive inflammation. Dehydroandrographolide (DA) possesses anti-inflammatory properties. However, the anti-inflammatory activities of DA in cholestatic liver injury remain unclear. METHODS: Mice were administered with DA by intraperitoneal injection after bile duct ligation (BDL) on day 1. Then mice were subjected to an ileocecal vein injection of lipopolysaccharide (LPS). Liver function markers, histology, pro-inflammatory cytokine levels, NF-κB activation and fibrosis formation were evaluated in BDL mice with LPS. LPS binding to primary Kupffer cells was examined by high-content cytometers. RESULTS: DA was shown to greatly lower initially higher than normal levels of alanine aminotransferase (ALT) and total bilirubin (TBIL) in the serum and liver of BDL mice with LPS. DA exerted hepatic protective effects that were also confirmed by prolonged survival of BDL mice with LPS. Liver histopathology showed reduced inflammatory cellular infiltration, bile duct proliferation, and biliary necrosis with DA treatment. Furthermore, DA reduced the expression levels of tumor necrosis factor (TNF)-α and interleukin (IL)-6 in liver tissue and plasma and showed decreased NF-κB activation in BDL mice with LPS. DA could prevent LPS binding to primary Kupffer cells in the normal liver and BDL mice liver. DA also suppressed LPS-stimulated inflammatory responses by blocking the interaction between LPS and TLR4 in primary Kupffer cells and human LX-2 cells, thereby inhibiting NF-κB activation. CONCLUSION: DA inhibition of inflammation against liver damage following BDL with LPS may be a promising agent for the treatment of cholestatic liver injury.

Second-order calibration serves as a remedial measure for the simultaneous determination of andrographolide and dehydroandrographolide in Andrographis paniculata and its preparations by HPLC without complete baseline separation.

In this work, high-performance liquid chromatography with diode array detection was applied for the simultaneous determination of andrographolide and dehydroandrographolide in Andrographis paniculata and its preparations. As a result of the incomplete baseline separation caused by complex backgrounds, the classical univariate calibration method failed to determine accurate contents of the analytes. On this occasion, chemometric second-order calibration based on the well-known alternating trilinear decomposition algorithm was then explored to serve as a post-experimental remedial tool to solve this problem. By using the intelligent "mathematical separation" of alternating trilinear decomposition, the peak areas of the analytes do not need to be directly measured and the predictive results become accurate. The contents of andrographolide and dehydroandrographolide were determined to be (7.95 ± 0.15) and (1.85 ± 0.02) µg/mL for Andrographis paniculata, (1.34 ± 0.01) and (5.53 ± 0.04) µg/mL for its preparations, which was in agreement with those obtained by a reference liquid chromatography with mass spectrometry method. This study showed the superiority of second-order calibration method over classical univariate calibration method for simultaneous determination of multi-analytes in complex samples. It also proved that second-order calibration may be a good choice for remedying incomplete baseline separation problem, with the accompanied reduction of experimental burden and toxic organic solvents as well as analysis time and cost.

The human mast cell line-1 cell membrane chromatography coupled with HPLC-ESI-MS/MS method for screening potentical anaphylactic components from chuanxinlian injection.

Chuanxinlian injection is a traditional Chinese medicine injection widely used in China to treat sore throat, cough and dysentery, although a high occurrence of severe adverse reactions has been reported in clinical practice in recent years. In the present study, a human mast cell line-1 cell membrane chromatography coupled with HPLC-ESI-MS/MS method was established to screen and identify potentical anaphylactic components in chuanxinlian injection, and the dehydroandrographolide was identified as a potential anaphylactic component. In vitro anaphylactic assay showed that intracellular Ca2+ concentration clearly increased under dehydroandrographolide (100 µm) treatment. ß-Hexosaminidase and histamine release in human mast cell line-1 cells were both markedly enhanced with increased concentrations of dehydroandrographolide, confirming the anaphylactic activity of dehydroandrographolide. The application for chuanxinlian injection in this study suggested that the developed human mast cell line-1 cell membrane chromatography coupled with HPLC-ESI-MS/MS system may be effective and rapid for screening the potentical anaphylactic components from complex samples.

Synthesis, structure-activity relationships and biological evaluation of dehydroandrographolide and andrographolide derivatives as novel anti-hepatitis B virus agents.

Dehydroandrographolide and andrographolide, two natural diterpenoids isolated from Andrographis paniculata possessed activity against HBV DNA replication with IC50 values of 22.58 and 54.07µM and low SI values of 8.7 and 3.7 in our random assay. Consequently, 48 derivatives of dehydroandrographolide and andrographolide were synthesized and evaluated for their anti-HBV properties to yield a series of active derivatives with lower cytotoxicity, including 14 derivatives against HBsAg secretion, 19 derivatives against HBeAg secretion and 38 derivatives against HBV DNA replication. Interestingly, compound 4e could inhibit not only HBsAg and HBeAg secretions but also HBV DNA replication with SI values of 20.3, 125.0 and 104.9. Furthermore, the most active compound 2c with SI value higher than 165.1 inhibiting HBV DNA replication was revealed with the optimal logP value of 1.78 and logD values. Structure-activity relationships (SARs) of the derivatives were disclosed for guiding the future research toward the discovery of new anti-HBV drugs.

Dehydroandrographolide alleviates rheumatoid arthritis by inhibiting neutrophil activation via LMIR3 in collagen induced arthritis rats.

Rheumatoid arthritis (RA) is an inflammatory disease which causes severe pain and disability. Neutrophils play essential roles in the onset and progression of RA; thus, inhibition of neutrophil activation is becoming a popular therapeutic strategy. Dehydroandrographolide has provided satisfactory outcomes in inflammatory diseases; however, its therapeutic effects and mechanism in RA are not fully understood. Leukocyte mono-immunoglobulin-like receptor 3 (LMIR3) is a negative regulator highly expressed in neutrophils. To determine whether dehydroandrographolide negatively regulated neutrophils activation via LMIR3, cytokines release and collagen-induced arthritis (CIA) rats were used in vitro and in vivo. Biacore, molecular docking analysis and molecular dynamics simulation were performed to prove the target of dehydroandrographolide. Moreover, the downstream signaling pathways of LMIR3 activation were analyzed by western blotting. Results showed that oral dehydroandrographolide administration of 2 mg/kg/day to CIA rats attenuated synovitis and bone and cartilage damage after the 28-day intervention, revealed using HE sections and micro-CT. Dehydroandrographolide significantly inhibited cytokine release and chemotaxis of LPS/TNF-α-activated neutrophils in vitro. Dehydroandrographolide inhibited neutrophils activation via binding to LMIR3. Moreover, dehydroandrographolide up-regulated the phosphorylation of SHP-1 and SHP-2, which are the essential kinases in the LMIR3 signaling pathways. This study revealed that dehydroandrographolide attenuated collagen-induced arthritis by suppressing neutrophil activation via LMIR3.

Dehydroandrographolide ameliorates doxorubicin-mediated cardiotoxicity by regulating autophagy through the mTOR-TFEB pathway.

The clinical application of doxorubicin (DOX) was limited by the serious cardiotoxicity. The traditional Chinese medicine Andrographis paniculata and its principal active component (Dehydroandrographolide, DA) have been well known for their diverse cardiovascular protective effects. However, the effects of DA on DOX-induced cardiotoxicity (DIC) were still unknown. In this study, we evaluated the effects and revealed the potential mechanisms of DA on DIC both in vivo and in vitro. The effects of DA on DIC were systematically assessed by echocardiography and histological assays. Western blot and flow cytometry were used to measure apoptosis of cardiomyocytes. Transmission electron microscopy and StubRFP-SensGFP-LC3 lentivirus were further used to assay autophagic flux. Our results showed that DA administration significantly improved cardiac function and attenuated DOX-induced cardiomyocyte apoptosis. Mechanically, DA restored autophagic flux and lysosome functions via inhibiting DOX-induced mTOR signal pathway activation and increasing the translocation of TFEB to the nucleus. However, activation of mTOR or knockdown of TFEB significantly inhibited the protective effects of DA against DIC by impacting lysosomal functions and autophagic flux. In conclusion, our results revealed that DA might be a potential cardioprotective agent against DIC.

Inflammatory Studies of Dehydroandrographolide: Isolation, Spectroscopy, Biological Activity, and Theoretical Modeling.

Dehydroandrographolide (DA) was isolated and experimentally characterized utilizing FT-IR, UV-Vis, and NMR spectroscopy techniques along with detailed theoretical modelled at the DFT/B3LYP-D3BJ/6-311 + + G(d,p) level of theory. Substantially, molecular electronic property investigations in the gaseous phase alongside five different solvents (ethanol, methanol, water, acetonitrile and DMSO) were comprehensively reported and compared with the experimental results. The globally harmonized scale (GHS), which is used to identify and label chemicals, was also utilized to demonstrate that the lead compound predicted an LD50 of 1190 mg/kg. This finding implies that consumers can safely consume the lead molecule. Notable impacts on hepatotoxicity, cytotoxicity, mutagenicity, and carcinogenicity were likewise found to be minimal to nonexistent for the compound. Additionally, in order to account for the biological performance of the studied compound, in-silico molecular docking simulation analysis was examined against different anti-inflammatory target of enzymes (3PGH, 4COX, and 6COX). From the examination, it can be inferred that DA@3PGH, DA@4COX, and DA@6COX, respectively, showed significant negative binding affinities of -7.2 kcal/mol, -8.0 kcal/mol, and - 6.9 kcal/mol. Thus, the high mean binding affinity in contrast to conventional drugs further reinforces these results as an anti-inflammatory agent.

Dehydroandrographolide facilitates M2 macrophage polarization by downregulating DUSP3 to inhibit sepsis-associated acute kidney injury.

BACKGROUND: Sepsis is perceived as lethal tissue damage and significantly increases mortality in combination with acute kidney injury (AKI). M2 macrophages play important roles in the secretion of anti-inflammatory and tissue repair mediators. We aimed to study the role of Dehydroandrographolide (Deh) in sepsis-associated AKI in vitro and in vivo through lipopolysaccharide (LPS)-induced macrophages model and cecal ligation and puncture-induced AKI mice model, and to reveal the mechanism related to M2 macrophage polarization. METHODS: Enzyme-linked immunosorbent assay kits were used to assess the levels of inflammatory factors. Expression of markers related to M1 macrophages and M2 macrophages were analyzed. Additionally, dual specificity phosphatase 3 (DUSP3) expression was tested. Cell apoptosis was evaluated by flow cytometry analysis and terminal-deoxynucleotidyl transferase-mediated nick end labeling staining. Moreover, renal histological assessment was performed by using hematoxylin and eosin staining. RESULTS: Deh reduced inflammation of THP-1-derived macrophages exposed to LPS. Besides, Deh induced the polarization of M1 macrophages to M2 and downregulated DUSP3 expression in THP-1-derived macrophages under LPS conditions. Further, DUSP3 overexpression reversed the impacts of Deh on the inflammation and M2 macrophages polarization of THP-1-derived macrophages stimulated by LPS. Additionally, human proximal tubular epithelial cells (HK-2) in the condition medium from DUSP3-overexpressed THP-1-derived macrophages treated with LPS and Deh displayed decreased viability and increased apoptosis and inflammation. The in vivo results suggested that Deh improved the renal function, ameliorated pathological injury, induced the polarization of M1 macrophages to M2, suppressed inflammation and apoptosis, and downregulated DUSP3 expression in sepsis-induced mice. CONCLUSION: Deh facilitated M2 macrophage polarization by downregulating DUSP3 to inhibit septic AKI.

Anti-inflammatory effect and pharmacokinetics of dehydroandrographolide, an active component of Andrographis paniculata, on Poly(I:C)-induced acute lung injury.

Acute lung injury (ALI) is a common and critical respiratory disorder caused by various factors, with viral infection being the leading contributor. Dehydroandrographolide (DAP), a constituent of the Chinese herbal plant Andrographis paniculata, exhibits a range of activities including anti-inflammatory, in vitro antiviral and immune-enhancing effects. This study evaluated the anti-inflammatory effects and pharmacokinetics (PK) profile of DAP in ALI mice induced by intratracheal instillation of Poly(I:C) (PIC). The results showed that oral administration of DAP (10-40 mg/kg) effectively suppressed the increase in lung wet-dry weight ratio, total cells, total protein content, accumulation of immune cells, inflammatory cytokines and neutrophil elastase levels in bronchoalveolar lavage fluid of PIC-treated mice. DAP concentrations, determined by an LC-MS/MS method, in plasma after receiving DAP (20 mg/kg) were unchanged compared to those in normal mice. However, DAP concentrations and relative PK parameters in the lungs were significantly altered in PIC-treated mice, exhibiting a relatively higher maximum concentration, larger AUC, and longer elimination half-life than those in the lungs of normal mice. These results demonstrated that DAP could improve lung edema and inflammation in ALI mice, and suggested that lung injury might influence the PK properties of DAP, leading to increased lung distribution and residence. Our study provides evidence that DAP displays significant anti-inflammatory activity against viral lung injury and is more likely to distribute to damaged lung tissue.

Potassium dehydroandrographolide succinate regulates the MyD88/CDH13 signaling pathway to enhance vascular injury-induced pathological vascular remodeling.

Pathological vascular remodeling is a hallmark of various vascular diseases. Previous research has established the significance of andrographolide in maintaining gastric vascular homeostasis and its pivotal role in modulating endothelial barrier dysfunction, which leads to pathological vascular remodeling. Potassium dehydroandrographolide succinate (PDA), a derivative of andrographolide, has been clinically utilized in the treatment of inflammatory diseases precipitated by viral infections. This study investigates the potential of PDA in regulating pathological vascular remodeling. The effect of PDA on vascular remodeling was assessed through the complete ligation of the carotid artery in C57BL/6 mice. Experimental approaches, including rat aortic primary smooth muscle cell culture, flow cytometry, bromodeoxyuridine (BrdU) incorporation assay, Boyden chamber cell migration assay, spheroid sprouting assay, and Matrigel-based tube formation assay, were employed to evaluate the influence of PDA on the proliferation and motility of smooth muscle cells (SMCs). Molecular docking simulations and co-immunoprecipitation assays were conducted to examine protein interactions. The results revealed that PDA exacerbates vascular injury-induced pathological remodeling, as evidenced by enhanced neointima formation. PDA treatment significantly increased the proliferation and migration of SMCs. Further mechanistic studies disclosed that PDA upregulated myeloid differentiation factor 88 (MyD88) expression in SMCs and interacted with T-cadherin (CDH13). This interaction augmented proliferation, migration, and extracellular matrix deposition, culminating in pathological vascular remodeling. Our findings underscore the critical role of PDA in the regulation of pathological vascular remodeling, mediated through the MyD88/CDH13 signaling pathway.

The effects and mechanisms of the anti-COVID-19 traditional Chinese medicine, Dehydroandrographolide from Andrographis paniculata (Burm.f.) Wall, on acute lung injury by the inhibition of NLRP3-mediated pyroptosis.

BACKGROUND: Dehydroandrographolide (Deh) from Andrographis paniculata (Burm.f.) Wall has strong anti-inflammatory and antioxidant activities. PURPOSE: To explore the role of Deh in acute lung injury (ALI) of coronavirus disease 19 (COVID-19) and its inflammatory molecular mechanism. METHODS: Liposaccharide (LPS) was injected into a C57BL/6 mouse model of ALI, and LPS + adenosine triphosphate (ATP) was used to stimulate BMDMs in an in vitro model of ALI. RESULTS: In an in vivo and in vitro model of ALI, Deh considerably reduced inflammation and oxidative stress by inhibiting NLRP3-mediated pyroptosis and attenuated mitochondrial damage to suppress NLRP3-mediated pyroptosis through the suppression of ROS production by inhibiting the Akt/Nrf2 pathway. Deh inhibited the interaction between Akt at T308 and PDPK1 at S549 to promote Akt protein phosphorylation. Deh directly targeted PDPK1 protein and accelerated PDPK1 ubiquitination. 91-GLY, 111-LYS, 126-TYR, 162-ALA, 205-ASP and 223-ASP may be the reason for the interaction between PDPK1 and Deh. CONCLUSION: Deh from Andrographis paniculata (Burm.f.) Wall presented NLRP3-mediated pyroptosis in a model of ALI through ROS-induced mitochondrial damage through inhibition of the Akt/Nrf2 pathway by PDPK1 ubiquitination. Therefore, it can be concluded that Deh may be a potential therapeutic drug for the treatment of ALI in COVID-19 or other respiratory diseases.

Application of 2H stable isotope labelling methodology and ultra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometry for the metabolite identification of dehydroandrographolide in rats.

Dehydroandrographolide (DA), one of the crucial diterpenoids of Andrographis paniculata (Burm.F.) Nees, which has been widely used clinically due to its excellent biological activities and pharmacological safety. Until now, various investigations about the biological activities, pharmacokinetic profiles, and in vitro metabolism of DA have been conducted. However, information about the in vivo biotransformation of DA was still not available. In this study, a rapid and reliable approach based on stable isotope labeling and UPLC-Q/TOF-MS was developed and applied for the first systematic research about the in vivo metabolism of DA. As a result, a total of 35 metabolites were identified in rat urine, bile, plasma, and feces samples after DA was orally administered at the dose of 95 mg/kg, and 33 of them were further verified based on stable isotope labeling. The major metabolic pathways for DA were hydroxylation, hydration, sulfonation, sulfate conjugation, and glucuronidation. Meanwhile, sulfonation, sulfate conjugation, and amino acids conjugation of DA were reported for the first time. This is the first systematic investigation of the in vivo metabolism of DA in rats, and the identification of these metabolites might provide scientific and reliable support for a full understanding of the metabolism of DA.

Comparison of pulmonary availability and anti-inflammatory effect of dehydroandrographolide succinate via intratracheal and intravenous administration.

Dehydroandrographolide succinate (DAS) injection, which was approved in China for the treatment of viral pneumonia and upper respiratory tract infections, is often off-label used for nebulization therapy to avoid the adverse drug reactions associated with the injection. However, the aerodynamic properties and pulmonary fate of nebulized DAS was largely uninvestigated. In this study, the main objectives were to evaluate the in vitro aerodynamic deposition profiles of nebulizer generated aerosols and comparatively investigate the local drug availability and anti-inflammatory efficacy of DAS between intratracheal and intravenous dosing. The in vitro evaluation of aerodynamic characteristics and droplet size distribution showed more than 50% aerosol particles with size being <5 µm, allowing the aerosols to reach the lower respiratory tract. Following intratracheal administration, the drug underwent pulmonary absorption into the bloodstream, rendering an absolute bioavailability of 47.3%. Compared to the intravenous delivery, the intratracheal administration dramatically increased the drug availability in the lung tissue in rats by more than 80-fold, leading to an improved and prolonged local anti-inflammatory efficacy in a lipopolysaccharide induced lung injury model in mice. The present results demonstrated that inhalation delivery of DAS is a convenient and effective alternative to intravenous injections.

Dehydroandrographolide inhibits mastitis by activating autophagy without affecting intestinal flora.

Mastitis can seriously damage the physical and mental health of lactating women. The use of antibiotics and anti-inflammatory drugs may damage the flora balance in lactating women. To alleviate mastitis in lactating women and reduce drug-induced damage to the flora, we found that dehydroandrographolide (Deh) has good anti-inflammatory and bacterial balance functions. In vivo, we found that Deh significantly inhibited the expression of MPO, IL6, IL-1ß, TNF-α, COX2 and iNOS and reduced pathological damage to the mammary gland. The feces in the control and Deh groups were collected and sequenced for 16S flora. The results showed that Deh did not change the primary intestinal microflora composition of the two groups. In vitro, our study showed that Deh significantly inhibited the expression of IL6, IL-1ß and TNF-α in the EpH4-Ev cell line. When an AMPK inhibitor was added, the anti-inflammatory effect of Deh was blocked. To further study the anti-inflammatory mechanism of Deh, we found that Deh significantly promoted autophagy through the phosphorylation of AMPK, Beclin and ULK1. In conclusion, our study found that Deh promoted autophagy and played an anti-inflammatory role by activating the AMPK/Beclin/ULK1 signaling pathway and did not affect intestinal flora.

14-Deoxy-11,12-didehydroandrographolide inhibits apoptosis in influenza A(H5N1) virus-infected human lung epithelial cells via the caspase-9-dependent intrinsic apoptotic pathway which contributes to its antiviral activity.

Influenza A virus (IAV) infection represents a global health challenge. Excavating antiviral active components from traditional Chinese medicine (TCM) is a promising anti-IAV strategy. Our previous studies have demonstrated that 14-deoxy-11,12-didehydroandrographolide (DAP), a major ingredient of a TCM herb called Andrographis paniculata, shows anti-IAV activity that is mainly effective against A/chicken/Hubei/327/2004 (H5N1), A/duck/Hubei/XN/2007 (H5N1), and A/PR/8/34 (H1N1) in vitro and in vivo. However, the underlying anti-IAV molecular mechanism of DAP needs further investigation. In the present work, we found that DAP can significantly inhibit the apoptosis of human lung epithelial (A549) cells infected with A/chicken/Hubei/327/2004 (H5N1). After DAP treatment, the protein expression levels of cleaved PARP, cleaved caspase-3, and cleaved caspase-9, and the activities of caspase-3 and caspase-9 in H5N1-infected A549 cells were all obviously downregulated. However, DAP had no inhibitory effect on caspase-8 activity and cleaved caspase-8 production. Meanwhile, the efficacy of DAP in reducing the apoptotic cells was lost after using the inhibitor of caspase-3 or caspase-9 but remained intact after the caspase-8 inhibitor treatment. Moreover, DAP efficiently attenuated the dissipation of mitochondrial membrane potential, suppressed cytochrome c release from the mitochondria to the cytosol, and decreased the protein expression ratio of Bax/Bcl-2 in the mitochondrial fraction. Furthermore, the silencing of caspase-9 reduced the yield of nucleoprotein (NP) and disabled the inhibitory ability of DAP in NP production in A549 cells. Overall results suggest that DAP exerts its antiviral effects by inhibiting H5N1-induced apoptosis on the caspase-9-dependent intrinsic/mitochondrial pathway, which may be one of the anti-H5N1 mechanisms of DAP.

Dehydroandrographolide Inhibits Osteosarcoma Cell Growth and Metastasis by Targeting SATB2-mediated EMT.

BACKGROUND: The 12-hydroxy-14-dehydroandrographolide (DP) is a predominant component of the traditional herbal medicine Andrographis paniculata (Burm. f.) Nees (Acanthaceae). Recent studies have shown that DP exhibits potent anti-cancer effects against oral and colon cancer cells. OBJECTIVE: This investigation examined the potential effects of DP against osteosarcoma cell. METHODS: A cell analyzer was used to measure cell viability. The cell growth and proliferation were performed by Flow cytometry and BrdU incorporation assay. The cell migration and invasion were determined by wound healing and transwell assay. The expression of EMT related proteins was examined by Western blot analysis. RESULTS: In this study, we found that DP treatment repressed osteosarcoma (OS) cell growth in a dose-dependent manner. DP treatment significantly inhibited OS cell proliferation by arresting the cell cycle at G2/M phase. In addition, DP treatment effectively inhibited the migration and invasion abilities of OS cells through wound healing and Transwell tests. Mechanistic studies revealed that DP treatment effectively rescued the epithelialmesenchymal transition (EMT), while forced expression of SATB2 in OS cells markedly reversed the pharmacological effect of DP on EMT. CONCLUSION: Our data demonstrated that DP repressed OS cell growth through inhibition of proliferation and cell cycle arrest; DP also inhibited metastatic capability of OS cells through a reversal of EMT by targeting SATB2. These findings demonstrate DP's potential as a therapeutic drug for OS treatment.

3-Dehydroandrographolide protects against lipopolysaccharide-induced inflammation through the cholinergic anti-inflammatory pathway.

Acute lung injury (ALI) is a deadly disease without effective chemotherapy, so far. Traditional Chinese medicine andrographis herba is frequently used in the treatment of respiratory diseases. In searching for natural anti-ALI components from andrographis herba, the activities of 3-dehydroandrographolide (3-DA), a new natural andrographolide product from andrographis herba were evaluated. In this study, murine macrophage RAW 264.7 cells and BALB/c mice were treated with LPS (lipopolysaccharide, 100 ng/ml in vitro; 3 mg/kg, intratracheal) to establish inflammation models. 3-DA attenuated the release of pro-inflammatory cytokines IL-6 and TNF-α, inhibited the degradation and phosphorylation of IκBα, and suppressed the nuclear translocation of NF-κB p65 as well as the phosphorylation of Akt at Ser473 in LPS-stimulated RAW 264.7 macrophage cells. Furthermore, 3-DA increased α7nAchR expression level and bound with α7nAchR. More importantly, the anti-inflammatory effects of 3-DA were counteracted in the presence of α7nAchR siRNA or methyllycaconitine (MLA, a α7nAchR specific inhibitor), suggesting that α7nAchR is a potential target in the anti-inflammatory effects of 3-DA. Besides, 3-DA significantly inhibited inflammation in LPS-induced ALI mice, which was associated with the decrease of lung water content and inflammatory cytokines, the inhibition of neutrophil and macrophage infiltration, and activation of the NF-κB/Akt signaling pathway. Moreover, these protective effects were attenuated by the treatment of MLA. Taken together, 3-DA alleviates LPS-induced inflammation via the cholinergic anti-inflammatory pathway in vitro and in vivo. These findings provide a rationale for the role of the cholinergic anti-inflammatory pathway in inflammation and the promising clinical application of 3-DA to treat ALI.

Protective effect of dehydroandrographolide on obstructive cholestasis in bile duct-ligated mice.

BACKGROUND: Dehydroandrographolide (DA) is the main contributor to the therapeutic properties of the medicinal plant Andrographis paniculata (AP). However, it is unknown whether DA has a hepatoprotective effect on obstructive cholestasis in mice and humans. METHODS: We administered DA to mice for 5 days prior to bile duct ligation (BDL) and for the 7 days. Liver function markers, liver histology and necrosis, compensatory responses of hepatocytes, liver fibrosis and the expression of hepatic fibrogenesis markers were evaluated in BDL mice and/or human LX-2 cells. RESULTS: Mice treated with DA demonstrated lower levels of serum alanine transarninase (ALT), milder liver damage, liver necrosis and fibrosis formation than in vehicle control with carboxymethylcellulose (CMC) mice after BDL. DA treatment also enhanced the Mrp3 expression of hepatocytes but not Mrp4 following BDL. Further, DA treatment in BDL mice significantly reduced liver mRNA and/or protein expression of Tgf-ß, Col1a1, α-Sma and Mmp2. This result was also supported by hydroxyproline analysis. The molecular mechanisms of DA treatment were also assessed in human hepatic stellate cell line (LX-2 cell). DA treatment significantly inhibited Tgf-ß-induced Col1a1, Mmp2 and α-Sma expression in human LX-2 cells. These data suggested that DA treatment reduced liver damage through development of a hepatic adaptive response and inhibition of the activation of HSCs, which led to a reduction in liver fibrosis formation in BDL mice. CONCLUSIONS: DA treatment protected against liver damage and fibrosis following BDL and might be an effective therapy for extrahepatic cholestasis due to bile duct obstruction.