Hydroxysafflower Yellow A Inhibits Vascular Adventitial Fibroblast Migration via NLRP3 Inflammasome Inhibition through Autophagy Activation.

Inflammation is closely associated with progression of vascular remodeling. The NLRP3 inflammasome is the key molecule that promotes vascular remodeling via activation of vascular adventitia fibroblast (VAF) proliferation and differentiation. VAFs have a vital effect on vascular remodeling that could be improved using hydroxysafflower yellow A (HSYA). However, whether HSYA ameliorates vascular remodeling through inhibition of NLRP3 inflammasome activation has not been explored in detail. Here, we cultured primary VAFs and analyzed the migration of VAFs induced by angiotensin II (ANG II) to determine the potential effects and mechanism of HSYA on VAF migration. The results thereof showed that HSYA remarkably inhibited ANG II-induced VAF migration, NLRP3 inflammasome activation, and the TLR4/NF-κB signaling pathway in a dose-dependent manner. In addition, it is worth noting that LPS promoted ANG II-induced VAF migration and NLRP3 inflammasome assembly, which could be significantly reversed using HSYA. Moreover, HSYA could be used to inhibit NLRP3 inflammasome activation by promoting autophagy. In conclusion, HSYA could inhibit ANG II-induced VAF migration through autophagy activation and inhibition of NLRP3 inflammasome activation through the TLR4/NF-κB signaling pathway.

Natural Deep Eutectic Solvents for the Extraction of Bioactive Steroidal Saponins from Dioscoreae Nipponicae Rhizoma.

In the present study, a simple and environmentally friendly extraction method based on natural deep eutectic solvents (NADESs) was established to extract four bioactive steroidal saponins from Dioscoreae Nipponicae Rhizoma (DNR). A total of twenty-one types of choline chloride, betaine, and L-proline based NADESs were tailored, and the NADES composed of 1:1 molar ratio of choline chloride and malonic acid showed the best extraction efficiency for the four steroidal saponins compared with other NADESs. Then, the extraction parameters for extraction of steroidal saponins by selected tailor-made NADES were optimized using response surface methodology and the optimal extraction conditions are extraction time, 23.5 min; liquid-solid ratio, 57.5 mL/g; and water content, 54%. The microstructure of the DNR powder before and after ultrasonic extraction by conventional solvents (water and methanol) and the selected NADES were observed using field emission scanning electron microscope. In addition, the four steroidal saponins were recovered from NADESs by D101 macroporous resin with a satisfactory recovery yield between 67.27% and 79.90%. The present research demonstrates that NADESs are a suitable green media for the extraction of the bioactive steroidal saponins from DNR, and have a great potential as possible alternatives to organic solvents for efficiently extracting bioactive compounds from natural products.

Mangiferin activates Nrf2 to attenuate cardiac fibrosis via redistributing glutaminolysis-derived glutamate.

Cardiac injury is followed by fibrosis, characterized by myofibroblast activation. Excessive deposition of extracellular matrix (ECM) impairs the plasticity of myocardium and results in myocardial systolic and diastolic dysfunction. Mangiferin is a xanthonoid derivative rich in plants mangoes and iris unguicularis, exhibiting the ability to ameliorate metabolic disorders. This study aims to investigate whether mangiferin attenuates cardiac fibrosis via redox regulation. The transverse aortic constriction (TAC) in mice induced cardiac fibrosis with impaired heart function. Oral administration of mangiferin (50 mg/kg, 4 weeks) inhibited myofibroblast activation with reduced formation of ECM. The impaired left ventricular contractive function was also improved by mangiferin. TGF-ß1 stimulation increased glutaminolysis to fuel intracellular glutamate pool for the increased demands of nutrients to support cardiac myofibroblast activation. Mangiferin degraded Keap1 to promote Nrf2 protein accumulation by improving its stability, leading to Nrf2 activation. Nrf2 transcriptionally promotes the synthesis of antioxidant proteins. By activating Nrf2, mangiferin promoted the synthesis of glutathione (GSH) in cardiac fibroblasts, likely due to the consumption of glutaminolysis-derived glutamate as a source. Meanwhile, mangiferin promoted the exchange of intracellular glutamate for the import of extracellular cystine to support GSH generation. As a result of redistribution, the reduced glutamate availability failed to support myofibroblast activation. In support of this, the addition of extracellular glutamate or α-ketoglutarate diminished the inhibitory effects of mangiferin on cardiac myofibroblast proliferation and activation. Moreover, cardiac knockdown of Nrf2 attenuated the cardioprotective effects of mangiferin in mice subjected to TAC. In conclusion, we demonstrated that activated myofibroblasts were sensitive to glutamate availability. Mangiferin activated Nrf2 and redistributed intracellular glutamate for the synthesis of GSH, consequently impairing cardiac myofibroblast activation due to decreased glutamate availability. These results address that pharmacological activation of Nrf2 could restrain cardiac fibrosis via metabolic regulation.

Correction to: Mangiferin suppresses endoplasmic reticulum stress in perivascular adipose tissue and prevents insulin resistance in the endothelium.

In the original publication of the article error has occurred in Fig. 1b.

Mangiferin suppresses endoplasmic reticulum stress in perivascular adipose tissue and prevents insulin resistance in the endothelium.

PURPOSE: Mangiferin is a naturally occurring glucosylxanthone with beneficial effects on glucose and lipid homeostasis. This study investigates the potential therapeutic effect of Mangiferin in perivascular adipose tissue (PVAT) and whether it contributes to regulating insulin action in the endothelium. METHODS: Palmitate challenge evoked ROS-associated endoplasmic reticulum stress (ER stress) and NLRP3 inflammasome activation in PVAT. The conditioned medium from PA-stimulated PVAT was prepared to induce endothelial insulin resistance, and improved endothelium-dependent vasodilation in response to insulin was detected in vitro and in vivo. RESULTS: Mangiferin treatment enhanced LKB1-dependent AMPK activity and suppressed ER stress with downregulation of TXNIP induction, leading to the inhibition of NLRP3 inflammasome activation evidenced by attenuated NLRP3 and cleaved caspase-1 expression as well as reduced IL-1ß secretion. Moreover, Mangiferin restored insulin-mediated Akt and eNOS phosphorylations with increased NO production, immunohistochemistry examination of adipocytes, and endothelial tissue in high-fat diet-fed mice also showed that oral administration of Mangiferin inhibited ER stress and NLRP3 induction in PVAT, and then effectively prevented insulin resistance in the vessel endothelium. CONCLUSIONS: Taken together, these results revealed that Mangiferin suppressed ER stress-associated NLRP3 inflammasome activation in PVAT through regulation of AMPK activity, which prevented endothelial insulin resistance. These findings suggested that the amelioration of PVAT dysfunction may be a therapeutic strategy for the prevention of endothelial insulin resistance.

Mangiferin protects mitochondrial function by preserving mitochondrial hexokinase-II in vessel endothelial cells.

Hexokinase-II (HK-II) confers protection against cell death and this study was designed to investigate the effect of mangiferin on the regulation of mitochondrial HK-II. In vessel endothelial cells, saturated fatty acid palmitate (PA) stimulation induced HK-II detachment from mitochondria due to cellular acidification. Mangiferin reduced lactate accumulation by improving pyruvate dehydrogenase activity, promoted Akt translocation to HK-II and prevented HK-II detachment from mitochondria. Knockdown of Akt2 diminished the protective effect of mangiferin on mitochondrial HK-II, confirming the role of Akt in the regulation of HK-II. Mangiferin prevented mitochondrial permeability transition pore opening, restored mitochondrial membrane potential and thereby protected cell from apoptosis. In high-fat diet fed mice, oral administration of mangiferin induced Akt phosphorylation, increased HK-II binding to mitochondria and resultantly protected vessel endothelial function, demonstrating its protective effect on endothelial integrity in vivo. This finding provided a novel strategy for the protection of mitochondrial function in the endothelium.

Mangiferin Protects DNase 2 Abundance via Nrf2 Activation to Prevent Cytosolic mtDNA Accumulation during Liver Injury.

SCOPE: Mitochondrial DNA (mtDNA) released into the cytosol serves as a member of damage-associated molecular patterns to initiate inflammatory responses. Mangiferin is a xanthonoid derivative, usually isolated from plants including mangoes and iris unguicularis. This study aims to investigate whether mangiferin prevents mtDNA accumulation in the cytosol with a focus on deoxyribonuclease 2 (DNase 2) protection from oxidative damage. METHODS AND RESULTS: Mangiferin administration effectively protects against hepatotoxicity in mice subjected to CCl4 challenge or bile duct ligation (BDL) surgery. Moreover, mangiferin activates nuclear factor erythroid 2-related factor (Nrf2)-antioxidant signaling, reduces cytosolic mtDNA accumulation, and suppresses Toll-like receptor 9 (TLR-9)/myeloid differentiation factor 88 (MyD88)-dependent inflammation in the liver. The study prepares hepatic mtDNA to stimulate hepatocytes, and finds that mangiferin protects DNase 2 protein abundance. mtDNA induces reactive oxygen species (ROS) production to promote DNase 2 protein degradation through oxidative modification, but mangiferin protects DNase 2 protein stability in a Nrf2-dependent manner. In hepatic Nrf2 deficiency mice, the study further confirms that Nrf2 induction is required for mangiferin to clear cytosolic mtDNA and block mtDNA-mediated TLR9/MyD88/nuclear factor kappa-B (NF-κB) inflammatory signaling cascades. CONCLUSION: These findings provide new insights into the role of mangiferin as a liver protecting agent, and suggest protection of DNase 2 as a novel therapeutic strategy for pharmacological intervention to prevent liver damage.

The complete chloroplast genome of Piptanthus nepalensis, a medicinal plant.

Piptanthus nepalensis (Hooker) Sweet is a semi deciduous or deciduous shrub belonging to the genus Piptanthus, Leguminosae. P. nepalensis has been used as a folk medicinal herb in Nepal and was cultivated all over the world as an ornamental plant. In the present study, we sequenced the entire genome of the chloroplast of P. nepalensis. The total length of the chloroplast genome in P. nepalensis is 152,195 bp, including a large single-copy region of 82,048 bp, a small single-copy region of 17,675 bp, and a pair of inverted repeats regions of 26,236 bp. The overall guanine-cytosine (GC) content of the genome was 36.7%. There are 131 genes in the chloroplast genome of P. nepalensis, including 85 protein-coding genes, 8 rRNA genes and 38 tRNA genes. Phylogenetic analysis showed that P. nepalensis is closely related to Maackia floribunda.

The complete chloroplast genome of Sophora davidii (Fabaceae) and its phylogenetic implications.

Sophora davidii (Franch.) Pavol. is a deciduous or evergreen shrubs belonging to the genus Sophora, Fabaceae. The roots of S. davidii have been traditionally used as a medicinal herb in China to clear internal heat, relieve sore throat, and reduce swelling. Here we sequenced the whole genome of the chloroplast of S. davidii. The complete length of the chloroplast genome in S. davidii is 153,584 bp, containing a large single-copy region of 83,930 bp, a small single-copy region of 15,008 bp, and a pair of inverted repeats regions of 25,823 bp. The total guanine-cytosine (GC) percentage of the chloroplast genome was 36.7%. A total of 131 genes were annotated from the chloroplast genome of S. davidii, including 85 protein-coding genes, 8 rRNA genes and 38 tRNA genes. The phylogenetic analysis showed that S. davidii is closely related to the other three species of genus Sophora.

Luteolin inhibits lysophosphatidylcholine-induced apoptosis in endothelial cells by a calcium/mitocondrion/caspases-dependent pathway.

Luteolin, a naturally occurring polyphenol flavonoid, has demonstrated some beneficial modulation toward the endothelium. This study aims to investigate the effects of luteolin on lysophosphatidylcholine (LPC)-induced apoptosis, a key event in the pathogenesis of atherosclerosis, in endothelial cells. Luteolin reduced not only LPC-induced cell death but also lactate dehydrogenase (LDH) leakage. Luteolin inhibition of LPC-induced apoptosis in endothelial cells demonstrated its protection against the cytotoxicity of LPC. LPC-induced apoptosis is characterized by a calcium-dependent mitochondrial pathway, involving calcium influx, activation of calpains, cytochrome C release and caspases activation. Luteolin reduced calcium influx. It also inhibited calpains activation and prevented the release of cytochrome C from mitochondrion. The inhibition of cytochrome C release by luteolin blocked the activation of caspase-3 and thus prevented subsequent endothelial cell apoptosis. These results suggested that luteolin inhibits LPC-induced apoptosis in endothelial cells through the blockage of the calcium-dependent mitochondrial pathway.

Mangiferin protects against alcoholic liver injury via suppression of inflammation-induced adipose hyperlipolysis.

Adipose dysfunction is closely associated with alcoholic liver disease. The impact of mangiferin on ethanol-induced liver injury and the probable underlying molecular mechanism has not been sufficiently addressed. In the present study, mice were subjected to a chronic plus a single binge ethanol feeding to induce liver injury. In addition, the differentiated adipocytes from primary mouse adipocytes were isolated and used for the mechanism studies. Our study demonstrated that mangiferin protects against ethanol induced adipose hyperlipolysis by restoring PDE3B stability, which is associated with activating the AMPK/TBK1 signaling and suppressing the noncanonical NF-κB activation, leading to the reduction of free fatty acid release and the amelioration of ethanol-induced liver injury. Our findings identify that mangiferin ameliorates alcoholic liver injury via suppression of inflammation-induced adipose hyperlipolysis, suggesting that mangiferin might be a potential effective agent for the management of alcoholic liver injury.

Overexpression of a sour jujube gene ZjPYR1, encoding a putative abscisic acid receptor, increases sensitivity of the stomata and roots to ABA in Arabidopsis thaliana.

The abscisic acid (ABA) receptor binds to ABA in plants and can activate the ABA signaling pathway to initiate stress resistance. Sour jujube (Ziziphus jujuba Mill. var. spinosa (Bunge) Hu ex H. Chou) is an economic tree crop in North China, which is strongly adapted to drought and salt stress. Here, we cloned and overexpressed the ZjPYR1 gene in Arabidopsis thaliana. After ABA treatment, the accumulation of ZjPYR1 increased significantly, suggesting that ABA may stabilize ZjPYR1 in the plants. Compared with the wild-type, the heterologous transgenic lines showed smaller stomatal openings under ABA treatment and a shorter root length and lower germination rate under ABA and salt treatment. Based on these results, we speculate that the overexpression of ZjPYR1 in A. thaliana effectively enhanced the stress resistance of the plants, and furthermore, that ZjPYR1 is a putative ABA receptor in sour jujube that increases plant adaptability to drought and salt stress. We report that ZjPYR1, like most ABA receptors in A. thaliana, is involved in mediating plant responses to ABA, such as stomatal closure and root length.

The aqueous extract of Gentianella acuta improves isoproterenol­induced myocardial fibrosis via inhibition of the TGF­ß1/Smads signaling pathway.

Gentianella acuta (G. acuta) is one of the most commonly used herbs in Chinese Mongolian medicine for the treatment of heart disease. Previously, it was found that G. acuta ameliorated cardiac function and inhibited isoproterenol (ISO)­induced myocardial fibrosis in rats. In this study, the underlying anti­fibrotic mechanism of G. acuta was further elucidated. Histopathological changes in the heart were observed by hematoxylin­eosin, Masson trichrome and wheat germ agglutinin staining. Relevant molecular events were investigated using immunohistochemistry and western blotting. The results revealed that G. acuta caused improvements in myocardial injury and fibrosis. G. acuta also inhibited collagens I and III and α­smooth muscle actin production in heart tissue. G. acuta downregulated the expression of transforming growth factor ß1 (TGF­ß1) and notably inhibited the levels of phosphorylation of TGF­ß receptors I and II. Furthermore, G. acuta caused downregulation of the intracellular mothers against decapentaplegic homolog (Smads)2 and 4 expression and inhibited Smads2 and 3 phosphorylation. The results further demonstrated that the mechanism underlying anti­myocardial fibrosis effects of G. acuta was based upon the suppression of the TGF­ß1/Smads signaling pathway. Therefore, G. acuta may be a potential therapeutic agent for ameliorating myocardial fibrosis.

Chelidonine enhances the antitumor effect of lenvatinib on hepatocellular carcinoma cells.

BACKGROUND: Lenvatinib is a newly approved molecular targeted drug for the treatment of advanced hepatocellular carcinoma (HCC). However, the high cost associated with this treatment poses a huge financial burden on patients and the entire public health system. Therefore, there is an urgent need to develop novel strategies that enhance the antitumor effect of lenvatinib. METHODS: The antitumor effects of chelidonine or/and lenvatinib on HCC cell lines MHCC97-H and LM-3 were examined using the 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2- H-tetrazolium bromide (MTT) assay. For the in-vivo investigation, the effect on subcutaneous or intrahepatic tumor growth in nude mice was also determined. The mRNA levels of epithelial mesenchymal transition (EMT)-related factors were examined through quantitative polymerase chain reaction or Western blot. RESULTS: In the present study, we found that treatment with chelidonine enhanced the apoptotic effect of lenvatinib on HCC cells and the in-vivo growth of HCC tumors in nude mice. Mechanistically, treatment with chelidonine increased the expression of epithelial indicator E-cadherin, whereas it decreased the expression of mesenchymal indicators N-cadherin and Vimentin. These findings suggest that chelidonine restricted the EMT in HCC cells. CONCLUSION: Chelidonine inhibits the process of EMT and enhances the antitumor effect of lenvatinib on HCC cells.

Roscovitine attenuates intimal hyperplasia via inhibiting NF-κB and STAT3 activation induced by TNF-α in vascular smooth muscle cells.

Roscovitine is a selective CDK inhibitor originally designed as anti-cancer agent, which has also been shown to inhibit proliferation in vascular smooth muscle cells (VSMCs). However, its effect on vascular remodeling and its mechanism of action remain unknown. In our study, we created a new intimal hyperplasia model in male Sprague-Dawley rats by trypsin digestion method, which cause to vascular injury as well as the model of rat carotid balloon angioplasty. Roscovitine administration led to a significant reduction in neointimal formation and VSMCs proliferation after injury in rats. Western blot analysis revealed that, in response to vascular injury, TNF-α stimulation induced p65 and STAT3 phosphorylation and promoted translocation of these molecules into the nucleus. p65 can physically associate with STAT3 and bind to TNF-α-regulated target promoters, such as MCP-1 and ICAM-1, to initiate gene transcription. Roscovitine can interrupt activation of NF-κB and reduce expression of TNF-α-induced proinflammatory gene, thus inhibiting intimal hyperplasia. These findings provide a novel mechanism to explain the roscovitine-mediated inhibition of intimal hyperplasia induced by proinflammatory pathways.

Mangiferin inhibits endoplasmic reticulum stress-associated thioredoxin-interacting protein/NLRP3 inflammasome activation with regulation of AMPK in endothelial cells.

BACKGROUND: Endothelial dysfunction is tightly associated with cardiovascular complications in diabetic patients. This study aims to investigate the effects of mangiferin on the regulation of endothelial homeostasis under endoplasmic reticulum stress (ER stress) conditions. RESULTS: High glucose (25 mmol/L) exposure induced ER stress and promoted ROS production in endothelial cells. Mangiferin effectively inhibited ER stress-associated oxidative stress by attenuating IRE1α phosphorylation and reducing ROS production. In response to ER stress, thioredoxin-interacting protein (TXNIP) expression increased, followed by NLRP3 inflammasome activation and increased IL-1ß secretion. Mangiferin treatment attenuated the expressions of TXNIP and NLRP3 and reduced IL-1ß and IL-6 production, demonstrating its inhibitory effects on TXNIP/NLRP3 inflammasome activation. NLRP3 inflammasome activation is responsible for mitochondrial cell death. Mangiferin restored the loss of the mitochondrial membrane potential (Δψm) and inhibited caspase-3 activity, and thereby protected cells from high glucose-induced apoptosis. Moreover, mangiferin inhibited ET-1 secretion and restored the loss of NO production when cells were exposed to high glucose. Mangiferin enhanced AMPK phosphorylation and AMPK inhibitor compound C diminished its beneficial effects, indicating the potential role of AMPK in its action. CONCLUSION: Our work showed the beneficial effects of mangiferin on the improvement of endothelial homeostasis and elucidated the molecular pathway through which mangiferin ameliorated endothelial dysfunction by inhibition of ER stress-associated TXNIP/NLRP3 inflammasome activation in endothelial cells. SIGNIFICANCE: These findings demonstrated the beneficial effects of mangiferin on the regulation of endothelial homeostasis and indicated its potential application in the management of diabetic cardiovascular complications.

Modified Si-Miao-San extract inhibits the release of inflammatory mediators from lipopolysaccharide-stimulated mouse macrophages.

ETHNOPHARMACOLOGICAL RELEVANCE: Modified Si-Miao-San (mSMS) is a prescription modified from Si-Miao-San which is an ancient Chinese prescription used to treat various ailments. AIM OF THE STUDY: Modified Si-Miao-San (mSMS) has been used for the treatment of infectious and inflammatory disorders in the clinic. This study was aimed to investigate its anti-inflammatory activity and underlying mechanism at cellular and molecular levels. MATERIALS AND METHODS: We stimulated RAW264.7 cells with Lipopolysaccharide (LPS) and observed effects of mSMS on the release of inflammatory mediators such as: tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), NO, and relative gene expressions. Meanwhile, we also investigated the modulation of mSMS in inflammatory signal transduction mediated through extracellular signal-regulated protein kinase (ERK) and nuclear factor-kappaB (NF-kappaB) pathway. RESULTS: Our findings demonstrated that mSMS significantly inhibited the excessive production of NO, TNF-alpha and IL-6 and the over expression of relative genes in LPS-stimulated macrophages. In addition, mSMS suppressed LPS-induced ERK1/2-phosphorylation and inhibited the activation of NF-kappaB by attenuation of I kappaB-alpha degradation. CONCLUSIONS: Our results suggest that the anti-inflammatory properties of mSMS might result from the inhibition of inflammatory mediators, such as NO, TNF-alpha and IL-6, via suppression of ERK and NF-kappaB dependent pathways.