(+)4-Cholesten-3-one promotes differentiation of neural stem cells into dopaminergic neurons through TET1 and FoxA2.

In this paper, we report the results of treating cells with an effective small molecule, (+)4-cholesten-3-one (PubChem CID: 91477), which can promote neural stem cell(NSC) differentiation into dopaminergic neurons. This study used rat neural stem cells stimulated with two different concentrations (7.8 µM and 78 µM) of (+)4-cholesten-3-one. Cell phenotypic analysis showed that (+)4-cholesten-3-one induced NSC differentiation into dopaminergic neurons, and the level of tyrosine hydroxylase(TH), which is specific for dopaminergic cells, was significantly increased compared with that of the drug-free control group. Furthermore, in this study, we found that this effect may be related to the transcription factor fork-head box a2 (FoxA2) and ten-eleven translocation 1 (TET1). The expression of TET1 and FoxA2 was upregulated after treatment with (+)4-cholesten-3-one. To verify the relationship between (+)4-cholesten-3-one and these genes, we found that the binding rate of TET1 and FoxA2 increased after the application of (+)4-cholesten-3-one, as confirmed by a coimmunoprecipitation (Co-IP) assay. With a small interfering RNA (siRNA) experiment, we found that only when Tet1 and Foxa2 were not silenced was the mRNA level of Th increased after (+)4-cholesten-3-one treatment. Taken together, these data show that (+)4-cholesten-3-one can promote the differentiation of NSCs into dopaminergic neurons by upregulating the expression of TET1 and FoxA2 and by increasing their binding. Thus, (+)4-cholesten-3-one may help address the application of neural stem cell replacement therapy in neurodegenerative diseases.

Wedelolactone facilitates Ser/Thr phosphorylation of NLRP3 dependent on PKA signalling to block inflammasome activation and pyroptosis.

OBJECTIVES: Wedelolactone exhibits regulatory effects on some inflammatory diseases. However, the anti-inflammatory mechanism of wedelolactone has not been entirely unravelled. Therefore, the present study focuses on investigating the mechanism of wedelolactone on NLRP3 inflammasome in macrophages and its influence on MSU-induced inflammation. MATERIALS AND METHODS: BMDM, J774A.1 and PMA-differentiated THP-1 macrophages were primed with LPS and then stimulated with ATP or nigericin or MSU crystal in the presence or absence of wedelolactone. The cell lysates and supernatants were collected to detect NLRP3 inflammasome components such as NLRP3, ASC and caspase 1, as well as pyroptosis and IL-1ß production. In addition, the anti-inflammatory effects of wedelolactone on MSU-induced peritonitis and arthritis mice were also evaluated. RESULTS: We found that wedelolactone broadly inhibited NLRP3 inflammasome activation and pyroptosis and IL-1ß secretion. Wedelolactone also block ASC oligomerization and speck formation. The inhibitory effects of wedelolactone were abrogated by PKA inhibitor H89, which also attenuated wedelolactone-enhanced Ser/Thr phosphorylation of NLRP3 at PKA-specific sites. Importantly, wedelolactone could abate MSU-induced IL-1ß production and neutrophils migration into peritoneal cavity, and reduced caspase 1 (p20) and IL-1ß expression in the joint tissue of MSU-induced arthritis. CONCLUSION: Our results indicate that wedelolactone promotes the Ser/Thr phosphorylation of NLRP3 to inhibit inflammasome activation and pyroptosis partly through potentiating PKA signalling, thus identifying its potential use for treating MSU-induced peritonitis and gouty arthritis.

Plastrum Testudinis Extracts Promote NSC Differentiation into Dopaminergic Neuron by Regulating the Interaction of TET1 and FoxA2.

In recent years, stem cells have gained much attention for the treatment of neurodegenerative diseases. However, inducing neural stem cell directionally differentiation is a difficult problem in the treatment of Parkinson's disease (PD) by stem cell therapy. Plastrum Testudinis (PT) can enhance the number of TH-positive neurons in the PD rat brain substantia nigra, but the underlying mechanism has not been clarified. Here, we aimed at further investigating the mechanism by which PT can promote NSC differentiation into dopaminergic neurons. A rat model of PD was used for detecting the effect of PT on the rat brain substantia nigra in vivo. The results showed the expressions of tyrosine hydroxylase (TH) and TET1 enzyme were increased after treatment with PT. Consequently, Plastrum Testudinis extracts (PTEs) were used for inducing NSC differentiation into dopaminergic neurons ex vivo. During differentiation of NSCs induced by PTE, TH expression was increased, with a concomitant increase in both TET1 and FoxA2. Next, we performed coimmunoprecipitation analysis to examine the interaction between TET1 protein and FoxA2 protein. Our results show that PTE can increase the binding rate of TET1 and FoxA2. Thus, our findings show that PTE can increase the efficiency of NSCs to directionally differentiate into dopaminergic neurons and provide experimental evidence for PT in the treatment of Parkinson's disease.

Anti-Inflammatory Effects of Shenfu Injection against Acute Lung Injury through Inhibiting HMGB1-NF-κB Pathway in a Rat Model of Endotoxin Shock.

Shenfu injection (SFI), a Chinese herbal medicine with substances extracted from Ginseng Radix et Rhizoma Rubra and Aconiti Lateralis Radix Praeparata, is widely used as an anti-inflammatory reagent to treat endotoxin shock in China. However, the mechanism of SFI in endotoxin shock remains to be illuminated. High mobility group box 1 (HMGB1), a vital inflammatory factor in the late stage of endotoxin shock, may stimulate multiple signalling cascades, including κB (NF-κB), a nuclear transcription factor, as well as tumour necrosis factor (TNF)-α and interleukin (IL)-1ß, among others in the overexpression of downstream proinflammatory cytokines. An investigation into the effects of SFI on the inhibition of the HMGB1-NF-κB pathway revealed the contribution of SFI to acute lung injury (ALI) in a rat model of endotoxin shock. To assess the anti-inflammatory activity of SFI, 5 ml/kg, 10 ml/kg, or 15 ml/kg of SFI was administered to different groups of rats following an injection of LPS, and the mean arterial pressure (MAP) at 5 h and the survival rate at 72 h were measured. 24 h after LPS injection, we observed pathological changes in the lung tissue and measured the mRNA expression, production, translocation, and secretion of HMGB1, as well as the expression of the NF-κB signal pathway-related proteins inhibitor of NF-κB (IκB)-α, P50, and P65. We also evaluated the regulation of SFI on the secretion of inflammatory factors including interleukin-1 beta (IL-1ß) and TNF-α. SFI effectively prevented the drop in MAP, relieved lung tissue damage, and increased the survival rate in the endotoxin shock model in dose-dependent manner. SFI inhibited the transcription, expression, translocation, and secretion of HMGB1, increased the expression of toll-like receptor (TLR4), increased the production of IκB-α, and decreased the levels of P65, P50, and TNF-α in the lung tissue of endotoxin shock rats in a dose-dependent manner. Furthermore, SFI decreased the secretion of proinflammatory cytokines TNF-α and IL-1ß. In summary, SFI improves the survival rate of endotoxin shock, perhaps through inhibiting the HMGB1-NF-κB pathway and thus preventing cytokine storm.

miR­134­5p/Foxp2/Syn1 is involved in cognitive impairment in an early vascular dementia rat model.

Forkhead box P2 (Foxp2) is a transcription factor involved in vocal learning. However, the number of previous studies that have investigated the role of Foxp2 in early vascular dementia (VD) is limited. The aim of the present study was to determine whether microRNA (miR)­134­5p/Foxp2 contributes to cognitive impairment in a chronic ischemia­induced early VD model. miR­134­5p was found to be significantly increased in the cortex in a rat VD model. Intracerebroventricular injection of miR­134­5p antagomir into VD rats prevented the loss of synaptic proteins and the development of cognitive impairment phenotypes. Histopathological analysis revealed that miR­134­5p aggravated cognitive impairment in VD rats through damage to cortical neurons and loss of synaptic proteins. Bioinformatics analysis predicted that miR­134­5p targets Foxp2 mRNA. Dual luciferase analysis and western blotting supported the prediction that miR­134­5p targets Foxp2. Furthermore, the silencing of Foxp2 significantly inhibited the effect of miR­134­5p on synaptic protein loss. Chromatin immunoprecipitation­quantitative polymerase chain reaction analysis indicated that Foxp2 binds to the synapsin I (Syn1) promoter at ­400/­600 bp upstream of the transcription start site. In conclusion, the miR­134­5p/Foxp2/Syn1 axis was found to contribute to cognitive impairment in a chronic ischemia­induced early VD model, which may enable the development of new therapeutic strategies for the prevention and treatment of VD.

miR­335 promotes stress granule formation to inhibit apoptosis by targeting ROCK2 in acute ischemic stroke.

Under harmful environmental conditions, stress granules (SGs), macromolecular aggregates that are associated with cell survival and death, are produced in the eukaryotic cytoplasm. However, whether and how microRNAs (miRNAs/miRs) modulate SG formation induced by acute ischemic stroke has not been investigated. In the present study, a rat model of middle cerebral artery occlusion (MCAO) was utilized and miRNA array profiling and reverse transcription­quantitative polymerase chain reaction were performed. The results revealed that miR­335 was downregulated during acute ischemic stroke, which was concomitant with reduced SG formation, enhanced apoptosis levels and increased Rho associated protein kinase 2 (ROCK2) expression. In the MCAO rat and serum­free cell models, miR­335 treatment upregulated SG formation, alleviated the ischemia­induced infarction, and decreased ROCK2 protein expression and apoptosis levels. By contrast, when compared with miR­335 treatment, the inhibition of miR­335 resulted in reduced SG formation and higher ROCK2 expression and apoptosis levels. Target prediction analysis and luciferase 3'­untranslated region reporter assay identified ROCK2 as the direct target of miR­335. Furthermore, ROCK2 silencing enhanced SG formation and attenuated the level of apoptosis in the serum­free cell model. In addition, ROCK2 silencing markedly inhibited the effect of miR­335 on SG formation and apoptosis levels. Unexpectedly, the phosphorylation of T­cell intracellular antigen­1 was significantly inhibited by miR­335 in the MCAO rat model, which provides a reasonable explanation for the promotional effect of miR­335 on SG formation by specifically targeting ROCK2. In conclusion, these results demonstrate that miR­335 promotes SG formation and inhibits apoptosis by reducing ROCK2 expression in acute ischemic stroke, which provides a possible therapeutic target for brain injury.

TET2-Mediated Spatiotemporal Changes of 5-Hydroxymethylcytosine During Organogenesis in the Late Mouse Fetus.

Genomic DNA demethylation is important for mammalian embryonic development and organ function. 5-Hydroxymethylcytosine (5hmC) is considered a novel epigenetic marker. Ten-eleven translocation (TET) enzymes convert 5-methylcytosine (5mC) to 5hmC. To explore the dynamic changes of epigenetic modifications during organogenesis in the late mouse fetus, the regional distribution and histological localization of 5hmC and TET enzymes was investigated by immunohistochemical method. The liver of mouse fetus gradually matured from embryonic day (E) 12.5 to E18.5.5mC was positive in developing liver at E16.5 and E18.5. 5hmC, TET2 and TET3 were strongly positive in hepatocytes and oval cells at E18.5. The small intestinal villi were formed at E16.5. The striate border and goblet cells appeared at E18.5. 5mC was detectable from E12.5 to E18.5. 5hmC and TET2 were positive in small intestine at E12.5, E14.5, and E18.5. The alveolar was formed at E18.5. 5mC and 5hmC were detectable from E12.5 to E18.5. Only TET2 was positive in the lung of the late Kunming mouse fetus. For vertebra, mesenchymal cells formed hyaline cartilage at E15.5 and then ossify at E16.5 and E18.8. 5mC, 5hmC, and TET2 were detectable in chondrocytes and osteocytes during the late Kunming mouse fetal; TET1 expressed from E14.5 to E16.5 and TET3 expressed in bone matrix at E18.5. In summary, TET2 was strongly expressed in liver, small intestinal, lung, and vertebra in the late Kunming mouse fetus. These findings suggested that TET2 may play a more critical role than TET1 and TET3 during organogenesis in the late stage of Kunming mouse embryo. Anat Rec, 302:954-963, 2019. © 2018 Wiley Periodicals, Inc.

Profiling the miRNA-mRNA-lncRNA interaction network in MSC osteoblast differentiation induced by (+)-cholesten-3-one.

BACKGROUND: Our previous study showed that (+)-cholesten-3-one (CN) has the potential to induce the osteoblastic differentiation of mesenchymal stem cells (MSCs). However, the roles of CN in targeting miRNA-mRNA-lncRNA interactions to regulate osteoblast differentiation remain poorly understood. RESULTS: A total of 77 miRNAs (36 upregulated and 41 downregulated) and 295 lncRNAs (281 upregulated and 14 downregulated) were significantly differentially expressed during CN-induced MSC osteogenic differentiation. Bioinformatic analysis identified that several pathways may play vital roles in MSC osteogenic differentiation, such as the vitamin D receptor signalling, TNF signalling, PI3K-Akt signalling, calcium signalling, and mineral absorption pathways. Further bioinformatic analysis revealed 16 core genes, including 6 mRNAs (Vdr, Mgp, Fabp3, Fst, Cd38, and Col1a1), 5 miRNAs (miR-483, miR-298, miR-361, miR-92b and miR-155) and 5 lncRNAs (NR_046246.1, NR_046239.1, XR_086062.1, XR_145872.1 and XR_146737.1), that may play important roles in regulating the CN-induced osteogenic differentiation of MSCs. Verified by the luciferase reporter, AR-S, qRT-PCR and western blot assays, we identified one miRNA (miR-298) that may enhance the osteogenic differentiation potential of MSCs via the vitamin D receptor signalling pathway. CONCLUSIONS: This study revealed the global expression profile of miRNAs and lncRNAs involved in the Chinese medicine active ingredient CN-induced osteoblast differentiation of MSCs for the first time and provided a foundation for future investigations of miRNA-mRNA-lncRNA interaction networks to completely illuminate the regulatory role of CN in MSC osteoblast differentiation.

miR­339­5p negatively regulates loureirin A­induced hair follicle stem cell differentiation by targeting DLX5.

Our previous study indicated that loureirin A induces hair follicle stem cell (HFSC) differentiation through Wnt/ß­catenin signaling pathway activation. However, if and how microRNAs (miRNAs/miRs) modulate loureirin A­induced differentiation remains to be elucidated. In the present study, HFSCs were separated from the vibrissae of rats and identified by CD34 and keratin, type 1 cytoskeletal (K)15 expression. Microarray­based miRNA profiling analysis revealed that miR­339­5p was downregulated in loureirin A­induced HFSC differentiation. miR­339­5p overexpression by transfection with miR­339­5p mimics markedly inhibited the expression of K10 and involucrin, which are markers of epidermal differentiation, whereas inhibition of miR­339­5p by miR­339­5p inhibitor transfection promoted the expression of K10 and involucrin. These results suggest that miR­339­5p is a negative regulator of HFSC differentiation following induction by loureirin A. These findings were confirmed by a luciferase assay. Homeobox protein DLX­5 (DLX5) was identified as a direct target of miR­339­5p. Furthermore, it was demonstrated that miR­339­5p inhibited DLX5. Overexpression of miR­339­5p by mimic transfection significantly inhibited protein Wnt­3a (Wnt3a) expression, while inhibition of miR­339­5p by inhibitor transfection significantly increased the expression of Wnt3a. Furthermore, small interfering RNA targeting DLX5 was transfected into HFSCs, and western blot analysis revealed that Wnt3a, involucrin and K10 expression was significantly downregulated. Taken together, these results suggest that miR­339­5p negatively regulated loureirin A­induced HFSC differentiation by targeting DLX5, resulting in Wnt/ß­catenin signaling pathway inhibition. This may provide a possible therapeutic target for skin repair and regeneration.

Paeonol attenuates acute lung injury by inhibiting HMGB1 in lipopolysaccharide-induced shock rats.

High-mobility group box 1 (HMGB1) is a highly conserved DNA-binding nuclear protein that facilitates gene transcription and the DNA repair response. However, HMGB1 may be released by necrotic cells as well as activated monocytes and macrophages following stimulation with lipopolysaccharide (LPS), interleukin-1ß (IL-1ß), or tumor necrosis factor-α (TNF-α). Extracellular HMGB1 plays a critical role in the pathogenesis of acute lung injury (ALI) through activating the nuclear transcription factor κB (NF-κB) P65 pathway, thus, it may be a promising therapeutic target in shock-induced ALI. Paeonol (Pae) is the main active component of Paeonia suffruticosa, which has been used to inhibit the inflammatory response in traditional Chinese medicine. We have proven that Pae inhibits the expression, relocation and secretion of HMGB1 in vitro. However, the role of Pae in the HMGB1-NF-κB pathway remains unknown. We herein investigated the role of Pae in LPS-induced ALI rats. In this study, LPS induced a marked decrease in the mean arterial pressure (MAP) and survival rate (only 25% after 72 h), and induced severe pathological changes in the lung tissue of rats, which was accompanied by elevated expression of HMGB1 and its downstream protein NF-κB P65. Treatment with Pae significantly improved the survival rate (>60%) and MAP, and attenuated the pathological damage to the lung tissue in ALI rats. Western blotting revealed that Pae also inhibited the total expression of HMGB1, NF-κB P65 and TNF-α in the lung tissue of ALI rats. Moreover, Pae increased the expression of HMGB1 in the nucleus, inhibited the production of HMGB1 in the cytoplasm, and decreased the expression of P65 both in the nucleus and cytoplasm of lung tissue cells in LPS-induced ALI rats. The results were in agreement with those observed in the in vitro experiment. These findings indicate that Pae may be a potential treatment for ALI through its repression of the HMGB1-NF-κB P65 signaling pathway.

Paeonol Reduces the Nucleocytoplasmic Transportation of HMGB1 by Upregulating HDAC3 in LPS-Induced RAW264.7 Cells.

Extracellular high mobility group box 1 (HMGB1) is a lethal pro-inflammatory mediator in endotoxin shock. Hyperacetylation of HMGB1, regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs), changes its subcellular localization and secretion to the extracellular matrix. Paeonol (2'-hydroxy-4'-methoxyacetophenone), one of the main active components of Paeonia suffruticosa, exerts anti-inflammatory effects. Our previous study demonstrated that Paeonol inhibited the relocation and secretion of HMGB1 in lipopolysaccharide (LPS)-activated RAW264.7 cells. However, it is still unclear whether Paeonol can regulate HATs/HDACs, which are responsible for the translocation of HMGB1 from nucleus to cytoplasm. To answer this question, P300 (a transcriptional coactivator with HATs) and HDAC3 were investigated using RT-qPCR and western blotting. The results showed that HMGB1 translocated from the nucleus to the cytoplasm, accompanied by upregulation of P300 and downregulation of HDAC3 in LPS-induced RAW264.7 cells. Paeonol, however, reversed the expression of P300 and HDAC3 significantly, suggesting that Paeonol may be involved in the acetylation of HMGB1 by regulating P300/HDAC3. Then, the effect of HDAC3 on the nucleocytoplasmic transportation of HMGB1 by HDAC3-SiRNA was evaluated. The results demonstrated that the inhibition of HDAC3 resulted in the nucleocytoplasmic translocation of HMGB1, with or without LPS stimulation. Moreover, Paeonol had no effect on the translocation of HMGB1 following ablation of HDAC3. These findings support the hypothesis that Paeonol can inhibit the translocation and secretion of HMGB1 in LPS-induced RAW264.7 cells by upregulating the expression of HDAC3. Paeonol may therefore be a valuable candidate as an HMGB1-targeting drug for inflammatory diseases via upregulation of HDAC3.

MiR-351 negatively regulates osteoblast differentiation of MSCs induced by (+)-cholesten-3-one through targeting VDR.

Our previous reports indicated that (+)-cholesten-3-one induces osteogenic differentiation of bone marrow mesenchymal stem cells (MSCs) by activating vitamin D receptor (VDR). However, whether and how miRNAs modulate osteogenic differentiation induced by (+)-cholesten-3-one have not been explored. In this study, miRNA array profiling and further validation by quantitative real-time PCR revealed that miR-351 was downregulated during (+)-cholesten-3-one-induced osteogenic differentiation of MSCs. Overexpression of miR-351 by miR-351 precursor transfection markedly inhibited the expression of osteoblast-specific genes, such as alkaline phosphatase (ALP), collagen type II, osteopontin (OPN), and runt-related transcription factor 2 (RUNX2), which consequently decreased a number of calcium mineralized nodules. Inhibition of miR-351 function by anti-miR-351 promoted expression of osteoblast-specific genes. Our results suggest that miR-351 is a negative regulator of osteoblast differentiation of MSCs induced by (+)-cholesten-3-one. Target prediction analysis tools and experimental validation by luciferase 3'UTR reporter assay identified VDR as a direct target of miR-351. miR-351 inhibited the expression of the VDR, which played a critical role in the control of osteogenic differentiation of MSCs. Importantly, overexpression of VDR significantly abolished the inhibitory effect of miR-351 on (+)-cholesten-3-one induced osteogenic differentiation. Taken together, our results demonstrate that miR-351 negatively regulates osteoblast differentiation of MSCs induced by (+)-cholesten-3-one through targeting VDR. These findings provid evidence that miR-351 can bea possible therapeutic target for bone repair and regeneration.

(+)-Cholesten-3-one induces osteogenic differentiation of bone marrow mesenchymal stem cells by activating vitamin D receptor.

In our previous reports, it was revealed that steroids in traditional Chinese medicine (TCM) have the therapeutic potential to treat bone disease. In the present study, an in vitro model of a vitamin D receptor response element (VDRE) reporter gene assay in mesenchymal stem cells (MSCs) was used to identify steroids that enhanced osteogenic differentiation of MSCs. (+)-cholesten-3-one (CN), which possesses a ketone group that is modified in cholesterol and cholesterol myristate, effectively promoted the activity of the VDRE promoter. Phenotypic cellular analysis indicated that CN induced differentiation of MSCs into osteogenic cells and increased expression of specific osteogenesis markers, including alkaline phosphatase, collagen II and Runt-related transcription factor 2. Furthermore, CN significantly increased the expression of osteopontin, the target of the vitamin D receptor (VDR), which indicated that CN may activate vitamin D receptor signaling. Over-expression of VDR or knockdown studies with VDR-small interfering RNA revealed that the pro-differentiation effects induced by CN required VDR. Furthermore, the present study determined that the C-terminal region of the VDR is responsible for the action of CN. Taken together, the present findings demonstrated that CN induced osteogenic differentiation of MSCs by activating VDR. The present study explored the regulation of stem cells by using a series of similar steroids and provided evidence to support a potential strategy for the screening of novel drugs to treat bone disease in the future.

Paeonol Inhibits Lipopolysaccharide-Induced HMGB1 Translocation from the Nucleus to the Cytoplasm in RAW264.7 Cells.

Transport of high-mobility group box 1 (HMGB1), a highly conserved non-histone DNA-binding protein, from the nucleus to the cytoplasm is induced by lipopolysaccharide (LPS). Secretion of HMGB1 appears to be a key lethal factor in sepsis, so it is considered to be a therapeutic target. Previous studies have suggested that paeonol (2'-hydroxy-4'-methoxyacetophenone), an active compound of Paeonia lactiflora Pallas, exerts anti-inflammatory effects. However, the effect of paeonol on HMGB1 is unknown. Here, we investigated the effect of paeonol on the expression, location, and secretion of HMGB1 in LPS-induced murine RAW264.7 cells. ELISA revealed HMGB1 supernatant concentrations of 615 ± 30 ng/mL in the LPS group and 600 ± 45, 560 ± 42, and 452 ± 38 ng/mL in cells treated with 0.2, 0.6, or 1 mM paeonol, respectively, suggesting that paeonol inhibits HMGB1 secretion induced by LPS. Immunohistochemistry and Western blotting revealed that paeonol decreased cytoplasmic HMGB1 and increased nuclear HMGB1. Chromatin immunoprecipitation microarrays suggested that HMGB1 relocation to the nucleus induced by paeonol might depress the action of Janus kinase/signal transducers and activators of transcription, chemokine, and mitogen-activated protein kinase pro-inflammatory signaling pathways. Paeonol was also found to inhibit tumor necrosis factor-α promoter activity in a dose-dependent manner. These results indicate that paeonol has the potential to be developed as a novel HMGB1-targeting therapeutic drug for the treatment of inflammatory diseases.

The protective effects of the supercritical-carbon dioxide fluid extract of Chrysanthemum indicum against lipopolysaccharide-induced acute lung injury in mice via modulating Toll-like receptor 4 signaling pathway.

The supercritical-carbon dioxide fluid extract of Chrysanthemum indicum Linné. (CFE) has been demonstrated to be effective in suppressing inflammation. The aim of this study is to investigate the preventive action and underlying mechanisms of CFE on acute lung injury (ALI) induced by lipopolysaccharide (LPS) in mice. ALI was induced by intratracheal instillation of LPS into lung, and dexamethasone was used as a positive control. Results revealed that pretreatment with CFE abated LPS-induced lung histopathologic changes, reduced the wet/dry ratio and proinflammatory cytokines productions (TNF-α, IL-1ß, and IL-6), inhibited inflammatory cells migrations and protein leakages, suppressed the levels of MPO and MDA, and upregulated the abilities of antioxidative enzymes (SOD, CAT, and GPx). Furthermore, the pretreatment with CFE downregulated the activations of NF-κB and the expressions of TLR4/MyD88. These results suggested that CFE exerted potential protective effects against LPS-induced ALI in mice and was a potential therapeutic drug for ALI. Its mechanisms were at least partially associated with the modulations of TLR4 signaling pathways.

Inhibitory effect of hydroxysafflor yellow a on mouse skin photoaging induced by ultraviolet irradiation.

Chronic exposure to ultraviolet (UV) irradiation is believed to be the major cause of skin damage that results in premature aging of the skin, so called photoaging, characterized by increases in skin thickness, formation of wrinkles, and loss of skin elasticity. UV induces damage to skin mainly by oxidative stress and collagen degradation. In this study, we examined the photo-protective effect of hydroxysafflor yellow A (HSYA), a major active chemical component isolated from Carthamus tinctorius L., by topical application on the skin of mice. Exposure of the dorsal depilated skin of mice to UV radiation four times a week for 10 weeks induced epidermal hyperplasia, elastin accumulation, collagen degradation, etc. HSYA at the doses of 50, 100, and 200 µg/mouse was topically applied immediately following each UV exposure. The effects of HSYA were evaluated by a series of tests, including macroscopic and histopathological evaluation of skin, pinch test, and redox homeostasis of skin homogenates. Results showed that the UV-induced skin damage was significantly improved after HSYA treatment, especially at doses of 100 and 200 µg/mouse. This protective effect is possibly related to the anti-oxidative property of HSYA and mediated by promoting endogenous collagen synthesis. This is the first study providing preclinical evidence for the protective effect of HSYA against photoaging.

ß-Asarone inhibits neuronal apoptosis via the CaMKII/CREB/Bcl-2 signaling pathway in an in vitro model and AßPP/PS1 mice.

ß-Asarone, an active component of the Acori graminei rhizome that has been used as traditional Chinese herb, has been reported to be capable of inhibiting neuronal apoptosis. However, the signaling mechanism underlying the inhibitory effect of ß-asarone has remained elusive. This study was aimed to investigate whether the CaMKII signaling pathway is involved in the ß-asarone mediated neuroprotection. Using PC12 cells and primary cultures of cortical neurons treated with amyloid-ß (Aß)(1-40) or Aß(1-42) peptide, we demonstrated that ß-asarone can protect PC12 cells and cortical neurons and inhibit neuronal apoptosis by activating the CaMKII-α/p-CREB/Bcl-2 pathway. Moreover, CaMKII-α overexpression enhanced the ß-asarone-induced p-CREB-Bcl-2 expression and anti-apoptotic effects. Interestingly, suppression of CaMKII-α by siRNA or a specific inhibitor can significantly reduce the ß-asarone-induced p-CREB and Bcl-2 expression and Aß(1-40) induced neuronal apoptosis in PC12 cells. AßPP/PS1 mice at the age of 3 months and age-matched wild-type mice were intragastrically administered ß-asarone (7 mg/kg/day, 21 mg/kg/day) or a vehicle daily for 4 months. ß-asarone improved cognitive function of the AßPP/PS1 mice and reduced neuronal apoptosis in the cortex of the AßPP/PS1 mice. A significant increase in CaMKII/CREB/Bcl-2 expression was observed in the cortex of the AßPP/PS1 mice treated with ß-asarone. In summary, our observations demonstrated that ß-asarone can inhibit neuronal apoptosis via the CaMKII/CREB/Bcl-2 signaling pathway in in vitro models and in AßPP/PS1 mice. Therefore, ß-asarone can be used as a potential therapeutic agent in the long-term treatment of Alzheimer's disease.

Muscone exerts neuroprotection in an experimental model of stroke via inhibition of the fas pathway.

Identifying small molecules that are neuroprotective against stroke injury will be highly beneficial for treatment therapies. A cell viability assay and gas chromatography-mass spectrometry were used to identify active small molecules in XingNaoJing, which is a well known Chinese medicine prescribed for the effective treatment of stroke. Studies have found that muscone is the active compound that prevents PC12 cell and cortical neuron damage following various injuries. Analysis of apoptosis indicated that muscone inhibited glutamate-induced apoptotic cell death of PC12 cells and cortical neurons. Fas and caspase-8 expression were upregulated following glutamate treatment in cortical neurons, and was markedly attenuated in the presence of muscone. Furthermore, muscone significantly reduced cerebral infarct volume, neurological dysfunction and inhibited cortical neuron apoptosis in middle cerebral artery occluded (MCAO) rats in a dose-dependent manner. Moreover, a significant decrease in Fas and caspase-8 expression in the rat cortex was observed in MCAO rats treated with muscone. Our results demonstrate that muscone may be a small active molecule with neuroprotective properties, and that inhibition of apoptosis and Fas is an important mechanism of neuroprotection by muscone. These findings suggest a potential therapeutic role for muscone in the treatment of stroke.

Houttuynia cordata inhibits lipopolysaccharide-induced rapid pulmonary fibrosis by up-regulating IFN-γ and inhibiting the TGF-ß1/Smad pathway.

This study aimed to explore the effect and mechanism of H. cordata vapor extract on acute lung injury (ALI) and rapid pulmonary fibrosis (RPF). We applied the volatile extract of HC to an RPF rat model and analyzed the effect on ALI and RPF using hematoxylin-eosin (H&E) staining, routine blood tests, a cell count of bronchoalveolar lavage fluid (BALF), lactate dehydrogenase (LDH) content, van Gieson (VG) staining, hydroxyproline (Hyp) content and the dry/wet weight ratio. The expression of IFN-γ/STAT(1), IL-4/STAT(6) and TGF-ß(1)/Smads was analyzed using ELISA, immunohistochemistry and western blotting methods. The active ingredients of the HC vapor extract were analyzed using a gas chromatograph-mass spectrometer (GC-MS), and the effects of the active ingredients of HC on the viability of NIH/3T3 and RAW264.7 cells were detected using an MTT assay. The active ingredients of the HC vapor extract included 4-terpineol, α-terpineol, l-bornyl acetate and methyl-n-nonyl ketone. The results of the lung H&E staining, Hyp content, dry/wet weight ratio and VG staining suggested that the HC vapor extract repaired lung injury and reduced RPF in a dose-dependent manner and up-regulated IFN-γ and inhibited the TGF-ß1/Smad pathway in vivo. In vitro, it could inhibit the viability of RAW264.7 and NIH/3T3 cells. It also dose-dependently inhibited the expression of TGF-ß1 and enhanced the expression of IFN-γ in NIH/3T3. The HC vapor extract inhibited LPS-induced RPF by up-regulating IFN-γ and inhibiting the TGF-ß1/Smad pathway.

Rapid pulmonary fibrosis induced by acute lung injury via a lipopolysaccharide three-hit regimen.

Based on the common characteristic of severe acute respiratory syndrome (SARS) and highly pathogenic avian influenza and the mechanism of inflammation and fibrosis, it is speculated that there should exist a fundamental pathological rule that severe acute lung injury (ALI)-induced rapid pulmonary fibrosis is caused by various etiological factors, such as SARS coronavirus, H5N1-virus, or other unknown factors, and also by lipopolysaccharide (LPS), the most common etiological factor. The investigation employed intratracheally, and intraperitoneally and intratracheally applied LPS three-hit regimen, compared with bleomycin-induced chronic pulmonary fibrosis. Inflammatory damage and fibrosis were evaluated, and the molecular mechanism was analyzed according to Th1/Th2 balance, Sma- and MAD-related proteins (Smads) and signal transducer and activator of transcriptions (STATs) expression. The results suggested that rapid pulmonary fibrosis could be induced by ALI via LPS three-hits. The period from 3-7 days in the LPS group was the first rapid pulmonary fibrosis stage, whereas the second fast fibrosis stage occurred on days 14-21. Th2 cell polarization, Smad4 and Smad7 should be the crucial molecular mechanism of ALI-induced rapid fibrosis. The investigation was not only performed to establish a new rapid pulmonary fibrosis model, but also to provide the elicitation for mechanism of ALI changed into the rapid pulmonary fibrosis.