1. Identifying Panaxynol From American Ginseng As A Suppressor Of Inflamed Macrophage-induced Cardiomyocyte Hypertrophy Via Nrf2 Up-regulation 2. Endothelial Atg7 Deficiency Enhances Skin Wound Healing Via Inflammation Up-regulation While Independent Of A

BackgroundGinseng is the root of genus Panax of the family Araliaceae. The English word Ginseng derives from a southern Chinese reading of the Chinese term "Ren Shen", meaning "man-herb," because of the humanoid shape of the root or rhizome of the plant. The botanical genus name of Panax ginseng was given by Carl Anton Meyer, a Russian botanist in 1843, and Panax is derived from Greek words "Pan" and "axos" meaning all and cure, respectively, which describes the traditional belief that ginseng has power to heal all aspects of the body. Ginseng has been used as a general tonic for thousands of years in Asian countries, and has become a popular herbal medicine world-wide. Recent research shows that regular use of ginseng is helpful in the treatment of Alzheimer’s disease, diabetes, aging, immune disorders, cancer and cardiovascular disease. However, the therapeutic efficacy of ginseng has not been established. The cellular and molecular mechanisms by which ginsengs induce pleiotropic biological actions remain largely unknown. Nevertheless, the public enthusiasms for using ginseng as an alternative medicine have been kept increasing, and it is estimated that ginseng might be the second top-selling herbal supplement in USA.Recently, the death of cardiovascular disease is increasing year by year; it is also the most important reason of death from all over the world. As we all know, cardiac hypotrophy in athletes and early stage of some disease helps increasing maximum cardiac output. However, if the causes of hypotrophy such as hypertension keep going, damage will become irreversible, including the maximum oxygen uptake decrease, maximum organic performance capacity decrease, and finally lead to pathologic cardiac hypertrophy and dysfunction.Nrf2 is a key transcription factor that binds to cis-acting enhancer sequence known as the antioxidant response element (ARE) with a core nucleotide sequence of 5’-RTGACnnnGC-3’to control the basal and inducible expression of more than 200 genes, which are functionally grouped into several categories including antioxidants, phase Ⅱ detoxifying enzymes, transcriptional factors, transporters, scavenger receptors, and chaperone proteins. As a result, Nrf2 appears to be a major transcription factor of cellular defense system against a variety of environmental or intrinsic insults in different organs including lung, liver, gastrointestinal tract, bladder, kidney, brain, skin, and ovary, and heart. Notably, Nrf2 signaling is cell type specific. The magnitude of Nrf2 activation seems to be functional relevant in specific settings: Constitutive activation of Nrf2 due the global knockout of its endogenous inhibitor, Keapl causes juvenile mortality, whereas activation of Nrf2 by knockdown of Keapl paratocially leads to either anti-diabetic or pro-diabetic phenotypes.We have demonstrated that knockout of Nrf2 results in earlier onset of cardiac maladaptive remodeling and dysfunction while cardiac specific over expression of Nrf2 is cardiac protective. The precise mechanisms of these discrepancies are presently unknown. Given the presence of a huge amount of Nrf2 activating small molecules that are naturally occurs or chemically synthesized, as well as the notion that some Nrf2 activators are capable of selectively driving Nrf2-mediated protection in target organs, Nrf2 has evolved to be an attractive drug target for the prevention and/or treatment of human diseases such as heart failure. However, a therapeutic Nrf2 activator remains to be established.Recently, we have found that American ginseng is capable of suppressing LPS-induced iNOS expression independent of NF-κB in macrophages and oxidative stress-mediated cell death in H9C2 cardiomyocytes via its ability to activate Nrf2. On the other hand, we have demonstrated that Nrf2 activation suppresses a selected set of pro-inflammatory cytokines including iNOS, monocyte chemotactic protein-1 (MCP-1), and macrophage inflammatory protein-1 beta (MIP-1β) while minimally regulating NF-κB activity and its downstream cytokine expression, such as interleulin-6 (IL-6), IL-1β, and tumor necrosis factor alpha (TNF-α) in macrophages. These results suggest that American ginseng contains substances which may activate Nrf2-mediated resolution of inflammatory responses in macrophages.Given the historically verified safety of American ginseng and the emerging evidence of American ginseng-induced Nrf2 activation for cardiac protection aforementioned, identifying the natural Nrf2 activating molecules from American ginseng may provide valuable insights into the development of novel Nrf2 activators to treat cardiac disease. Therefore, in the present study, we performed a bioassay based fractionation of American ginseng, aiming at the isolation of Nrf2 activating single compounds which are capable of specifically driving Nrf2-mediated health benefit in the heart. We found that American ginseng-derived panaxynol is a potent activator of Nrf2 in macrophages and enables Nrf2-mediated resolution of inflammatory responses in inflamed macrophages, which cause cardiomyocyte death and hypertrophy. These results suggest that selective activation of Nrf2 by panaxynol may constitute a promising approach to combat cardiac disease.Objectives1. To establish dose depend toxicity of American Ginseng (Am. G), find out safe dose for each extract.2. To screen the anti-inflammation function of all the extracts of Am. G.3. To discuss role of Am. G in hypotrophy.4. To discuss mechanisms of Am. G in hypotrophy, role of Nrf2 in protecting cardiomyocytes from hypotrophy.Materials and methods1. Purchased Nrf2 gene knockout mice (Nrf2-/-).2. Purchased all extracts of Am. G, including Crude, Hexane and Panaxynol.3. Culture RAW264.7 cells, L929 cells, mouse bone marrow derived macrophages as well as H9c2 cells to do the following experiments.4. Dual-luciferase reporter assay system.5. [3H] Leucine uptake assay.6. Protein expression detected by immunofluorescence.7. Protein expression detected by Western blot.8. Gene expression detected by Q-PCR.9. Statistical analysis.All data are expressed as X±SD, the differences between groups were compared using ANOVA analysis. SPSS statistical software was used for statistical analysis and P<0.05 was considered statistically significant.Results1. Bioassay-based fractionating anti-inflammatory components of American ginseng in macrophages.Cytotoxicity assay showed that the crude extract of American ginseng, hexane fraction, and water fraction at doses of 500 μg/ml and 1000μg/ml are toxic, and butanol fraction exerted a minor toxic effect, while the others at doses tested are non-toxic in RAW264.7 macrophages. Thus, we determined the effect of the crude extract and other fractions a non-toxic dose of 100 μg/ml on further study.2. Some Am. G extracts inhibit LPS-induced iNOS expressionWe found that LPS-induced iNOS expression was suppressed only by the crude extract, hexane fraction and its further purification panaxynol fraction. These results suggest that the inhibitory substances of American ginseng on iNOS expression are most likely derived from the hexane fraction.3. Pro-inflammatory cytokine expression in LPS-inflamed RAW264.7 cells treated by crude, hexane and panaxynol.American ginseng crude extract suppressed LPS-induced expression of iNOS, monocyte chemotactic protein 1 (MCP-1), and macrophage inflammatory protein-1 (MIP-1) p and minimally regulated LPS-induced expression of interleukin-1 (IL-1) β, IL-6, and tumor necrosis factor (TNF) a. Like American ginseng crude extract, the hexane fraction and panaxynol inhibited the expression of iNOS, MCP-1, and MIP-1 β and did not affect the expression of IL-1 β, and IL-6, and TNF-a in LPS-inflamed cells. These results demonstrate that panaxynol suppresses the expression of a selective set of pro-inflammatory cytokines in inflamed macrophages.4. Characterizing the potentials of American ginseng-derived components on Nrf2 activation in macrophages.To determine panaxynol as an Nrf2 activator contributing to American ginseng-induced resolution of inflammatory responses in macrophages, we, first examined the effects of American ginseng crude extract, hexane fraction, and panaxynol on Nrf2 activation in Raw264.7 cells. All of them upregulated Nrf2 protein expression and nuclear translocation while activating the expression of NQO1, a typical Nrf2 target gene. Next, we measured the effects of American ginseng crude extract, hexane fraction, and panaxynol on the ARE-Luc activity which indicates Nrf2-driven transcription as well as the Neh2-Luc activity that reflects the amount of Nrf2 escaped from Keapl-mediated degradation. We found that American ginseng crude extract enhanced both ARE-luc and Neh2-Luc activities, whereas the hexane fraction and panaxynol activated ARE-Luc activity without affecting Neh2-Luc activity. 5. An essential role of Nrf2 in mediating panaxynol-induced suppression of inflammatory responses in macrophagesAmerican ginseng crude extract and panaxynol inhibited iNOS expression in LPS-inflamed bone marrow-derived macrophages of WT mice. However, the inhibitory effects of American ginseng crude extract and panaxynol were attenuated and blocked respectively in bone marrow-derived macrophages of Nrf2-/- mice.6. Effects of American ginseng crude extract, the hexane fraction, and panaxynol on inflamed macrophage-mediated cell death and hypertrophic growth in cardiomyocytes.Conditioned medium of unstimulated RAW264.7 cells slightly increased cell death and induced hypertrophic growth in H9C2 cardiomyocytes, whereas conditioned medium of LPS-inflamed RAW264.7 cells caused dramatic augments of the cell death and hypertrophic growth. However, the RAW264.7 cell-mediated H9C2 cell death and hypertrophy were strongly suppressed by the treatment of American ginseng crude extract, the hexane fraction, and panaxynol in Raw264.7 cells.7. Effects of American ginseng crude extract, the hexane fraction, and panaxynol on inflamed Nrf2 knockdown macrophage-mediated cell death and hypertrophic growth in cardiomyocytes.After establishing the efficacy of Nrf2 siRNA in knocking down of Nrf2 in RAW264.7 cells, we determined the effects of conditioned medium of scramble siRNA and Nrf2 siRNA transfected RAW264.7 cells treated with or without American ginseng crude extract and panaxynol. As expected, the knockdown of Nrf2 in RAW264.7 cells wiped out the inhibitory effects of American ginseng crude and panaxynol on RAW264.7 cell conditioned medium-induced H9C2 cell death and hypertrophic growth.Conclusions1. Pro-inflammatory cytokine expression was down-regulated in LPS-inflamed RAW264.7 cells treated by crude, hexane and panaxynol.2. American ginseng crude extract, the hexane fraction, and panaxynol protected inflamed macrophage-mediated cell death and hypertrophic growth in cardiomyocytes.3. Panaxynol protected macrophage-mediated cell death and hypertrophic growth in cardiomyocytes via Nrf2.4. Protection of panaxynol to inflammation disappeared in Nrf2 knockout macrophages, as well as the protection to cardiomyocytes in Nrf2 knockdown RAW264.7 cells.BackgroundSkin wound healing is a complex series of reactions and interaction amongdermal cells and humoral mediators which are characterized by several overlappingphases including immediate humoral factor release, inflammatory responses,angiogenesis, proliferation, reepithelialization, and remodeling. The recruitment ofinflammatory cells at the early phase is important for the transition into the later phase,while angiogenesis, a process of new blood vessel formation characterized by thesprouting of pre-existing blood vessels, is believed to be one of the major biologicalresponses supporting cutaneous wound healing.In the early phase, homostasis and inflammation accrued around the wound.Collagen activates clotting cascade and a lot of cytokines and growth factoraccumulated around the wound, such as EGF, FGF, IFNs, ILs. Because of thesecytokines and growth factors, monocytes were recruited from nearby tissue and bloodinto the injury, and then transform in to macrophagy. Neutrophils arrive shortly aftermonocytes, and clean cellular debris and invading bacteria. Sufficient macrophagesactivation is impartment for the transition from inflammatory phase into proliferativephase.During the angiogenesis, angioblasts differentiate towards endothelial cells, thesecells proliferate into cords and then lumen occurs. Attracted by VEGF, endothelialcells become mobile and invasive. Top cell spearhead new filopodia out of the vessels,and probe the environment guidance cues. Stalk cells followed by, which proliferatingcells to support sprout elongation and helping form cord as well as lumen. Blood flowinitiate, which helping vessels start perfusion and maturation, finally supply oxygenand nutrition to the wound.Autophagy (Greek for "self-eating") is an evolutionarily conserved pathway thattarget cytoplasmic components to the lysosome for degradation. Autophagy has beenclassified into three different types depending on the means by which the target isdelivered into the lysosome: macroautophagy, microautophagy and chaperone-mediated autophagy. Macroautophagy (hereafter referred as autophagy) is thought tobe the major type of autophagy, and it is critical for the clearance of dysfunctionalproteins and damaged organelles in the cell thus maintaining physiologicalhomeostasis. Moreover, autophagy generally acts as an important adaptive mechanismfor cell survival.During the whole process, Atg7 as an ubiquitin-aetivating enzyme. helpingforming Atgl2-Atg5-Atgl6L1 complex and helping LC3 I conjugated to PE. 1,Atgl2 actived by Atg7, an E1 ubiquitin-activating enzyme, and then conjugated toAtgl0, an E2 ubiquitin-conjugating enzyme. By the help of E1 and E2, Atgl2 couldconjugated to Atg5 and then to Atgl6; 2, E1 (Atg7), E2 (Atg3) and E3(Atg 12-Atg5 -Atg 16L 1 complex) helped phophatidylethanolamine (PE) conjugated toLC3 I, the lapidated form of LC3 is called LC3 II, plays an important role duringphagophore elongation.It has been documented that regardless of nutrient states, the inhibition ofangiogenesis activates autophagy in endothelial cells. While hypoxia-inducedangiogenesis is increased also by the autophagy deficiency via the geneticdownregulation of Beclin 1, an initiator of autophagy in endothelial cells. Moreover,the activation of autophagy by a derivate of natural product magnolol (Ery5) inendothelial cells is also capable of inhibiting angiogenesis. On the other hand,angiogenesis is enhanced by the activation of autophagy due to the nutritiondeprivation or Atg5 overexpression in endothelial cells.However, deregulated autophagy is detrimental to the cell, particularly in somepathophysiological settings, thereby contributing to the pathogenesis of numerousdiseases. The precise pathophysiological relevance of autophagy remains poorlyunderstood. Because the overactivation of autophagy is linked to cell death, it hasbeen proposed that the excessive autophagy-indueed endothelial cell death may be theattributing mechanism to the observed autophagy-mediated suppression ofangiogenesis. Nevertheless, a potential role of endothelial autophagy-mediatedangiogenesis in skin wound healing is unclear.Therefore, in the present study, we investigated the impact of endothelialautophagy impairment on angiogenesis in skin wound healing using geneticallymanipulated mice with endothelial cell-specific knockout of Atg7, an E-1 likeubiquitin-activing enzyme which activates autophagy by inducting autophagosomeformation. To discuss the impact of endothelial Atg7 knockout during skin woundhealing, from inflammatory responses, angiogenesis and ite protential machenisms.Objectives 1. To breed mice with endothelial cell-specific knockout of Atg7, and check th eftieney of knockout. 2. To estabilish mouse skin wound healing model, and analysis data. 3. To investigate role of endothelial cell-specific knockout of Atg7 during skinwound healing, and discuss the mechanisms of speeding up the healing rate inAtg7Ec’/" mice.Materials and methods 1. Purchased Floxed-Atg7F/F mice and Cdh5-Cre+/+ mice. Cross bred these twostrains to get Atg7EC-/- mice as well as the other three kinds of control mice. 2. Checked endothelial Atg7 knockout effiency by Western blot. 3. Analysed wound healing rate by ImageJ software. 4. Analysed morphological parameter by H&E staining 5. Protein expression detected by immunofluorescence. 6. [3H] Thymidine uptake assay to check endothelial cells proliferation. 7. Statistical analysis. All data are expressed as X+SD, the differences betweengroups were compared using ANOVA analysis. SPSS statistical software was used forstatistical analysis and P<0.05 was considered statistically significant.Results1. Generation of Atg7 endothelial cell-specific knockout (Atg7Ec-/-) mice.Atg7 endothelial cell-specific knockout (Atg7Ec4") mice were generated bycrossing floxed-Atg7 (Atg7F/F) mice with Cdh5-Cre+/+ mice. There was anapproximately 50% decrease in the protein expression of Atg7 in lung endothelialcells freshly isolated from heterozygote Atg7EC mice, and an about 70% decreaseof Atg7 expression in lung endothelial cells freshly isolated from homozygoteAtg7Ec4" mice, compared to wide type (WT) mice. Meanwhile, LC3-I was markedlyincreased while the LC3-Ⅱ expression was significantly reduced in the isolated cells.The alternations of LC3-Ⅰ and Ⅱ were well co-related to the genotypes of Atg7knockout mice. Thus, the conditional Atg7Ec-/- mice were successfully generated.2. Histological morphologies in WT, Atg7F/F, Cdh5-Cre+/+ and Atg7EC-/- mice.The Atg7 endothelial cell-specific knockout did not induce apparent changes inhistological morphologies of endothelial cell-enriched organs including thoracic aorta,carotid artery, lung, and kidney, suggesting that loss of Atg7 in endothelial cells has aminimal impact on the development of major organ systems.3. The impact of flox and Cre gene inserts on skin wound healing at the basallevel.Since any genetic manipulation potentially affects some pathophysiologicalprocesses, we determined whether the flox and Cre insertions per se have any impacton skin wound healing in mice. Compared with the WT control, the healing offull-thickness skin excisional wounds was delayed in Atg7F/F mice at day 8 and day 10after skin injury, but not in Cdh5-Cre+/+ mice. These results indicate that the floxinserts flanked the exon 14 ofAtg7 gene slightly suppress skin wound healing.Although the underlying mechanism remains to be determined, these results revealedthat Atg7F/F mice have to be used as the control for the experiments using Atg7EC-/-mice.4. The effect of Atg7 endothelial cell-specific knockout on skin wound healingTo investigate the effect of Atg7 endothelial cell-specific knockout on skinwound healing, we examined the healing processes of full-thickness skin excisionalwounds in Atg7EC-/- mice using Atg7F/F mice as the control. Macroscopic analyses oftime-matched Atg7EC-/- versus Atg7F/F control wounds showed that wound closure ismarkedly accelerated at the indicated time points during the wound repair in Atg7EC-/-mice, with a mean of 85% wound closure achieved by day 10 in Atg7EC-/- mice anda mean of 70% wound closure in Atg7F/F mice.5. Skin tissue histological morphology anlysis in Atg7F/F and Atg7EC-/- mice.Wound reepithelialization as measured by length of epithelial tongue andepithelial gap at day 3 after wounding was significantly faster in Atg7EC-/- micecompared with Atg7F/F mice. However, there was no significant difference ingranulation tissue formation between Atg7EC-/- and Atg7F/F mice at 3 day after woundinjury.6. The effect of Atg7 endothelial cell-specific knockout on angiogenesis in skinwound healing and the effect of loss of Atg7 on endothelial cell proliferation in aprimary culture of HUVECs.To determine whether Atg7EC-/- impairs angiogenesis in skin wound healing,microscopic analyses of CD31 (an established endothelial biomarker) staining inwounded skin were performed. There was no significant difference of CD31 positivecells between Atg7F/F and Atg7EC-/- mice, suggesting that loss of Atg7 in endothelialcells hardly affects angiogenesis in skin wound healing. To further confirm theobservation, we examined the effect of knocking down of Atg7 on endothelial cellproliferation in a primary culture of HUVECs. The transfection ofAtg7 siRNAknocked down more than 90% of Atg7 protein expression in HUVECs; however, thisAtg7 knockdown did not exert detectable changes in [3H]-Thymidine uptake inHUVECs. These results suggest that autophagy impairment in endothelial cells byknockout of Atg7 may not affect endothelial cell proliferation and angiogenesis.7. The effect of Atg7 endothelial cell-specific knockout on the recruitment ofmacrophages and lyrnphocytes in skin wound healingBecause an early inflammatory phase appears to influence wound closure, wedetermined whether Atg7EC-/- leads to an alternation of inflammatory responses atearly phase of skin wound healing. The recruitments of macrophages andlymphocytes at 3 days after skin injury were increased in Atg7Ec-/- mice compared toAtg7F/F mice, indicating that Atg7EC-/- enhances inflammatory responses at the earlyphase of skin wound healing.Conclusions1. Atg7 protein expression is dramatictly decreased in Atg7EC-/- mice, comparedto wide type; however, it did not induce apparent changes in histologicalmorphologies of endothelial cell-enriched organs.2. Atg7F/F mice have to be used as the control for the experiments using Atg7EC-/-mice, because the flox inserts Atg7 gene slightly suppress skin wound healing.3. Wound healing rate was increased in Atg7EC-/- mice compared to Atg7F/F mice.4. Loss of Atg7 in endothelial cells hardly affects angiogenesis in skin woundhealing, and Atg7 knockdown did not exert detectable changes in [3H]-Thymidineuptake in HUVECs.4. Atg7EC-/- enhances inflammatory responses at the early phase of skin woundhealing, which is the key points to accelerate wound healing rate.