Mechanism of Shenfu Injection in Treating Ischemic Stroke Elucidated using Network Pharmacology and Experimental Validation.

BACKGROUND: Shenfu injection was derived from the classical Chinese medicine formula 'Shenfu decoction', which was widely used in the treatment of cardiovascular and cerebrovascular diseases in clinical practice. OBJECTIVES: Predict the main active ingredients, core targets, and related signaling pathways of Shenfu injection in the treatment of ischemic stroke. METHODS: Databases were used to collect the active ingredients and target information of Shenfu injection; GO and KEGG pathway enrichment analyses were performed using the David database. The effects of Shenfu injection on core targets were verified using molecular docking and in vivo experiments. RESULTS: The predicted results identified 44 active ingredients and 635 targets in Shenfu injection, among which 418 targets, including TNF, IL-6, MAPK1, and MAPK14, were potential targets for the treatment of ischemic stroke. Molecular docking revealed that the active ingredients had good binding to IL-6, MAPK1, and MAPK14. In vivo experiments demonstrated that Shenfu injection significantly improved the pathological damage due to ischemic stroke, promoted the expression of tight junction proteins, and inhibited MMP-2 and MMP-9 expressions, thereby reducing BBB permeability. Animal experiments revealed that Shenfu injection could inhibit p38、JNK and ERK phosphorylation. CONCLUSIONS: Mechanism of Shenfu injection in treating ischemic stroke may be via inhibition of the inflammatory factors levels and protecting the BBB, thereby warranting subsequent studies and highlighting its potential as a reference for new drug development.

Single-cell RNA sequencing unveils Lrg1's role in cerebral ischemia‒reperfusion injury by modulating various cells.

BACKGROUND AND PURPOSE: Cerebral ischemia‒reperfusion injury causes significant harm to human health and is a major contributor to stroke-related deaths worldwide. Current treatments are limited, and new, more effective prevention and treatment strategies that target multiple cell components are urgently needed. Leucine-rich alpha-2 glycoprotein 1 (Lrg1) appears to be associated with the progression of cerebral ischemia‒reperfusion injury, but the exact mechanism of it is unknown. METHODS: Wild-type (WT) and Lrg1 knockout (Lrg1-/-) mice were used to investigate the role of Lrg1 after cerebral ischemia‒reperfusion injury. The effects of Lrg1 knockout on brain infarct volume, blood‒brain barrier permeability, and neurological score (based on 2,3,5-triphenyl tetrazolium chloride, evans blue dye, hematoxylin, and eosin staining) were assessed. Single-cell RNA sequencing (scRNA-seq), immunofluorescence, and microvascular albumin leakage tests were utilized to investigate alterations in various cell components in brain tissue after Lrg1 knockout. RESULTS: Lrg1 expression was increased in various cell types of brain tissue after cerebral ischemia‒reperfusion injury. Lrg1 knockout reduced cerebral edema and infarct size and improved neurological function after cerebral ischemia‒reperfusion injury. Single-cell RNA sequencing analysis of WT and Lrg1-/- mouse brain tissues after cerebral ischemia‒reperfusion injury revealed that Lrg1 knockout enhances blood‒brain barrier (BBB) by upregulating claudin 11, integrin ß5, protocadherin 9, and annexin A2. Lrg1 knockout also promoted an anti-inflammatory and tissue-repairing phenotype in microglia and macrophages while reducing neuron and oligodendrocyte cell death. CONCLUSIONS: Our results has shown that Lrg1 mediates numerous pathological processes involved in cerebral ischemia‒reperfusion injury by altering the functional states of various cell types, thereby rendering it a promising therapeutic target for cerebral ischemia‒reperfusion injury.

Therapeutic effects and mechanism of Atractylodis rhizoma in acute lung injury: Investigation based on an Integrated approach.

Acute lung injury (ALI) is characterized by an excessive inflammatory response. Atractylodes lancea (Thunb.) DC. is a traditional chinese medicine with good anti-inflammatory activity that is commonly used clinically for the treatment of lung diseases in China; however, its mechanism of against ALI is unclear. We clarified the therapeutic effects of ethanol extract of Atractylodis rhizoma (EEAR) on lipopolysaccharide (LPS)-induced ALI by evaluation of hematoxylin-eosin (HE) stained sections, the lung wet/dry (W/D) ratio, and levels of inflammatory factors as indicators. We then characterized the chemical composition of EEAR by ultra-performance liquid chromatography and mass spectrometry (UPLC-MS) and screened the components and targets by network pharmacology to clarify the signaling pathways involved in the therapeutic effects of EEAR on ALI, and the results were validated by molecular docking simulation and Western blot (WB) analysis. Finally, we examined the metabolites in rat lung tissues by gas chromatography and mass spectrometry (GC-MS). The results showed that EEAR significantly reduced the W/D ratio, and tumor necrosis factor-α (TNF-α), interleukin-1 beta (IL-1ß), interleukin-6 (IL-6) levels in the lungs of ALI model rats. Nineteen components of EEAR were identified and shown to act synergetically by regulating shared pathways such as the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)-protein kinase B (AKT) signaling pathways. Ferulic acid, 4-methylumbelliferone, acetylatractylodinol, atractylenolide I, and atractylenolide III were predicted to bind well to PI3K, AKT and MAPK1, respectively, with binding energies < -5 kcal/mol, although only atractylenolide II bound with high affinity to MAPK1. EEAR significantly inhibited the phosphorylation of PI3K, AKT, p38, and ERK1/2, thus reducing protein expression. EEAR significantly modulated the expression of metabolites such as D-Galactose, D-Glucose, serine and D-Mannose. These metabolites were mainly concentrated in the galactose and amino acid metabolism pathways. In conclusion, EEAR alleviates ALI by inhibiting activation of the PI3K-AKT and MAPK signaling pathways and regulating galactose metabolism, providing a new direction for the development of drugs to treat ALI.

Deep-fried Atractylodes lancea rhizome alleviates spleen deficiency diarrhea-induced short-chain fatty acid metabolic disorder in mice by remodeling the intestinal flora.

ETHNOPHARMACOLOGICAL RELEVANCE: Atractylodes lancea (Thunb.) DC. is a Chinese herb that has been commonly used to treat spleen-deficiency diarrhea (SDD) in China for over a thousand years. However, the underlying mechanism of its antidiarrheal activity is not fully understood. AIM OF THE STUDY: The antidiarrheal effects of the ethanol extract of deep-fried A. lancea rhizome (EEDAR) due to spleen deficiency induced by folium sennae (SE) were determined on the regulation of the short-chain fatty acid (SCFA) metabonomics induced by the intestinal flora. MATERIALS AND METHODS: The effects of EEDAR on a SE-induced mouse model of SDD were evaluated by monitoring the animal weight, fecal water content, diarrhea-grade rating, goblet cell loss, and pathological changes in the colon. The expression of inflammatory factors (tumor necrosis factor [TNF]-α, interleukin [IL]-1ß, IL-6, IL-10), aquaporins (AQP3, AQP4, and AQP8), and tight junction markers (ZO-1, occludin, claudin-1) in colon tissues were determined using quantitative polymerase chain reaction and western blotting. SCFA metabonomics in the feces of mice treated with EEDAR was evaluated using gas chromatography-mass spectrometry. Furthermore, 16S rDNA sequencing was used to determine the effect of EEDAR on the intestinal flora of SDD mice, and fecal microbiota transplantation (FMT) was used to confirm whether the intestinal flora was essential for the anti-SDD effect of EEDAR. RESULTS: Treatment with EEDAR significantly improved the symptoms of mice with SDD by inhibiting the loss of colonic cup cells, alleviating colitis, and promoting the expression of AQPs and tight junction markers. More importantly, the effect of EEDAR on the increase of SCFA content in mice with SDD was closely related to the gut microbiota composition. EEDAR intervention did not significantly improve intestinal inflammation or the barrier of germ-free SDD mice, but FMT was effective. CONCLUSION: EEDAR alleviated SE-induced SDD in mice, as well as the induced SCFA disorder by regulating the imbalance of the intestinal microbiota.

Protective Effects of Atractylodis lancea Rhizoma on Lipopolysaccharide-Induced Acute Lung Injury via TLR4/NF-κB and Keap1/Nrf2 Signaling Pathways In Vitro and In Vivo.

Acute lung injury (ALI) is a syndrome caused by an excessive inflammatory response characterized by intractable hypoxemia both inside and outside the lung, for which effective therapeutic drugs are lacking. Atractylodis rhizoma, a traditional Chinese medicine, has excellent anti-inflammatory and antiviral properties in addition to protecting the integrity of the cellular barrier. However, few studies of Atractylodis rhizoma for the treatment of ALI have been published, and its mechanism of action remains unclear. In the present study, the chemical composition of the ethanolic extract of Atractylodis rhizoma (EEAR) was initially clarified by high performance liquid chromatography (HPLC), after which it was studied in vivo using a lipopolysaccharide (LPS)-induced ALI rat model. Treatment with EEAR significantly reduced the lung wet/dry (W/D) ratio, neutrophil infiltration, and malondialdehyde (MDA) and myeloperoxidase (MPO) formation, and enhanced superoxide dismutase (SOD) and glutathione (GSH) depletion in rats with ALI, thereby improving lung barrier function and effectively reducing lung injury. In addition, EEAR significantly reduced histopathological changes, decreased the expression of inflammatory factors (such as tumor necrosis factor-α (TNF-α), interleukin-1 beta (IL-1ß), inducible nitric oxide synthase (INOS), and cyclooxygenase-2 (COX-2)), and inhibited the activation of the NF-κB signaling pathway, thus reducing inflammation. In addition, EEAR was found to also reduce oxidative stress in ALI by upregulating the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and its downstream proteins heme oxygenase-1 (HO-1) and NADPH quinone acceptor oxidoreductase 1 (NQO-1). EEAR also reduced LPS-induced inflammatory factor expression in THP-1 cells in vitro by inhibition of the NF-κB signaling pathway, and reduced damage from lipopolysaccharide (LPS)-induced oxidative stress in THP-1 cells by promoting the expression of Nrf2 and its downstream targets HO-1 and NQO-1, the molecular mechanism of which was consistent with in vivo observations. Therefore, we conclude that EEAR attenuates oxidative stress and inflammatory responses via TLR4/NF-κB and Keap1/Nrf2 signaling pathways to alleviate LPS-induced ALI, suggesting that Atractylodis rhizoma is a potential drug candidate for the treatment of ALI.

Preventive effect of Atractylodis Rhizoma extract on DSS-induced acute ulcerative colitis through the regulation of the MAPK/NF-κB signals in vivo and in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Atractylodes lancea (Thunb.) DC. is traditionally used as a folk medicine for treating gastrointestinal diseases in China. Nevertheless, the effect and mechanisms of its anti-inflammatory activity on dextran sodium sulfate (DSS)-induced ulcerative colitis (UC) have not yet been fully investigated. AIM OF THE STUDY: This study aimed to explore the therapeutic effect and underlying molecular mechanisms of Ethanolic Extract of Atractylodis Rhizoma (EEAR) on DSS-induced UC mice and LPS-induced RAW264.7 cells. MATERIALS AND METHODS: The EEAR was obtained and then analyzed by HPLC analysis. The protective effect of EEAR on DSS-induced UC was evaluated by weight loss, disease activity index (DAI) score, spleen index, goblet cell loss, colon length shortening, myeloperoxidase (MPO) activity and pathological changes. The level of inflammatory cytokines were detected by immunohistochemistry (IHC) and RT-PCR analysis. The expressions of the tight junction (TJ, such as ZO-1, Occludin) proteins and the target proteins in mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) were determined by western blotting analysis. RESULTS: EEAR significantly attenuated the symptoms of UC, suppressed the colon MPO activity, and increased the goblet cell loss. In addition, EEAR could significantly increase the expression of TJs in UC mice. Meanwhile, EEAR treatment could reduce the levels of inflammatory cytokines and inhibit the phosphorylation of MAPK and NF-κB signaling pathways in UC mice and in LPS-induced RAW264.7 cells. CONCLUSION: Our results indicated that EEAR ameliorated DSS-induced UC by inhibiting the inflammatory response and maintaining the intestinal barrier function via modulation of MAPK/NF-κB pathways, thus, EEAR might be a promising therapeutic candidate for UC therapy.

Atractylodin Attenuates Dextran Sulfate Sodium-Induced Colitis by Alleviating Gut Microbiota Dysbiosis and Inhibiting Inflammatory Response Through the MAPK Pathway.

In this study, we investigated the therapeutic effects and mechanism of atractylodin (ATL) on dextran sulfate sodium (DSS)-induced ulcerative colitis in mice. We found that atractylodin could significantly reverse the effects of DSS-induced ulcerative colitis, such as weight loss, disease activity index score; shorten the colon length, and reverse the pathological changes in the colon of mice. Atractylodin could inhibit the activation of colonic macrophages by inhibiting the MAPK pathway and alleviate intestinal inflammation in the mouse model of ulcerative colitis. Moreover, it could protect the intestinal barrier by inhibiting the decrease of the tight junction proteins, ZO-1, occludin, and MUC2. Additionally, atractylodin could decrease the abundance of harmful bacteria and increase that of beneficial bacteria in the intestinal tract of mice, effectively improving the intestinal microecology. In an LPS-induced macrophage model, atractylodin could inhibit the MAPK pathway and expression of the inflammatory factors of macrophages. Atractylodin could also inhibit the production of lactate, which is the end product of glycolysis; inhibit the activity of GAPDH, which is an important rate-limiting enzyme in glycolysis; inhibit the malonylation of GAPDH, and, thus, inhibit the translation of TNF-α. Therefore, ours is the first study to highlight the potential of atractylodin in the treatment of ulcerative colitis and reveal its possible mechanism.

Atractylodes oil alleviates diarrhea-predominant irritable bowel syndrome by regulating intestinal inflammation and intestinal barrier via SCF/c-kit and MLCK/MLC2 pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Atractylodes lancea (Thunb.) DC. is a widely used traditional herb that is well known for treating spleen deficiency and diarrhea. According to traditional Chinese medicine (TCM) theory, diarrhea-predominant irritable bowel syndrome (IBS-D) is caused by cold and dampness, resulting in diarrhea and abdominal pain. Nevertheless, the effect and mechanism of Atractylodes on IBS-D are still unclear. AIM OF THE STUDY: This study was designed to confirm the therapeutic effect of Atractylodes lanceolata oil (AO) in a rat model of IBS-D, and to determine the mechanisms by which AO protects against the disease. MATERIALS AND METHODS: The chemical components in AO were determined using gas chromatography-mass spectrometry (GC-MS). The expression levels of 5-hydroxytryptamine (5-HT), vasoactive intestinal peptide (VIP), and surfactant protein (SP) in serum and colon tissue were measured using enzyme-linked immunosorbent assay (ELISA). Reverse transcription-polymerase chain reaction (RT-PCR), western blotting (WB), immunohistochemistry (IHC), and immunofluorescence (IF) were used to elucidate the mechanism of action of AO toward inflammation and the intestinal barrier in a rat model of IBS-D. RESULTS: The 15 chemical substances of the highest concentration in AO were identified using GC-MS. AO was effective against IBS-D in the rat model, in terms of increased body weight, diarrhea grade score, levels of interleukin-10 (IL-10), aquaporin 3 (AQP3), and aquaporin 8 (AQP8), and reduced fecal moisture content, levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), 5-HT, VIP, and SP, while also reducing intestinal injury, as observed using hematoxylin-eosin (HE) staining. In addition, the results indicated that AO increased the mRNA and protein expression levels of stem cell factor (SCF) and c-kit and enhanced the levels of zonula occludens-1 (ZO-1) and occludin, as well as decreased the levels of myosin light chain kinase (MLCK) and inhibited the phosphorylation of myosin light chain 2 (p-MLC2). CONCLUSIONS: AO was found to be efficacious in the rat model of IBS-D. AO inhibited the SCF/c-kit pathway, thereby reducing inflammation and protecting against intestinal barrier damage via the MLCK/MLC2 pathway.

Endothelial Conditional Knockdown of NMMHC IIA (Nonmuscle Myosin Heavy Chain IIA) Attenuates Blood-Brain Barrier Damage During Ischemia-Reperfusion Injury.

BACKGROUND AND PURPOSE: In ischemic stroke, breakdown of the blood-brain barrier (BBB) aggravates brain damage. Endothelial detachment contributes to BBB disruption and neurovascular dysfunction, but its regulation in stroke has yet to be clarified. We investigated the function of NMMHC IIA (nonmuscle myosin heavy chain IIA) in the endothelium on BBB breakdown and its potential mechanisms. METHODS: Endothelial conditional knockdown NMMHC IIA (Myh9ECKD) was constructed in vivo and in vitro, and its role was explored in middle cerebral artery occlusion/reperfusion-injured mice and oxygen-glucose deprivation/reoxygenation-injured brain microvascular endothelial cells. The degree of brain injury was analyzed using staining (2,3,5-triphenyltetrazolium chloride, hematoxylin, and eosin) and electron microscopy. BBB breakdown was investigated with leakage of Evans Blue dye and expression of TJs (tight junctions) and MMP (matrix metallopeptidase)-2/9. Transcriptomics for enrichment analysis was adopted to explore the potential downstream signaling pathways of NMMHC IIA involved in middle cerebral artery occlusion/reperfusion-induced BBB dysfunction. RESULTS: NMMHC IIA expression was upregulated in endothelial cells after cerebral ischemia/reperfusion injury. Myh9ECKD mice exhibited improvement in endothelial barrier hyperpermeability and TJs integrity stimulated by cerebral ischemia/reperfusion. Blebbistatin (NMMHC II inhibitor) treatment exerted the same effect. Transcriptomics showed that NMMHC IIA was involved in regulating various BBB-related genomic changes in the middle cerebral artery occlusion/reperfusion model, and NMMHC IIA was confirmed to significantly modulate Hippo and peroxisome proliferator-activated receptor gamma/nuclear factor-kappa B signaling pathways, which are closely related to BBB damage. CONCLUSIONS: Our findings provide some new insights into how NMMHC IIA contributes to maintaining the integrity of the cerebral endothelial barrier. NMMHC IIA could be a potential therapeutic target for ischemic stroke.

Pharmacogenetics of tamoxifen therapy in Asian populations: from genetic polymorphism to clinical outcomes.

BACKGROUND: Compared with western countries, Asian breast cancer patients have unique pathological and biological characteristics. Most of them are premenopausal women with HR positive. Tamoxifen as the first-line drug for premenopausal women with HR+ is involved in multiple enzymes and transporters during metabolizing and transporting process. Variants that cause decreased or inactive gene products leading to abnormal responses in tamoxifen therapy have well been studied in western countries, whereas such information is much less reported in Asian populations. OBJECTIVE: In order to elucidate the relationship between genetic variants and tamoxifen-induced individual drug reactions in different Asian populations and further identify genotypes/phenotypes with potential therapeutic significance. METHODS: We reviewed the frequencies of genetic variants in major enzymes and transporter genes involved in the metabolism and transport of tamoxifen across Asian populations as well as significant correlations between genotypes/metabolic phenotypes and metabolites concentrations or BC clinical outcomes. RESULTS: Significant inter-ethnic differences in allele frequencies was found among Asian populations, such as CYP2D6*4, *10, *41, CYP2C9*2, ABCB1 C3435T and SLCO1B1*5, and CYP2D6*10/*10 is the most common genotype correlated with adverse clinical outcomes. Moreover, we summarized the barriers and controversies of implementing pharmacogenetics in tamoxifen therapy and concluded that more population-specific pharmacogenetic studies are needed in the future. CONCLUSION: This review revealed more systematic pharmacogenomics of genes involved in the metabolism and transport besides CYP2D6, are required to optimize the genotyping strategies and guide the personalized tamoxifen therapy in Asian populations.

Treatment of Spleen-Deficiency Syndrome With Atractyloside A From Bran-Processed Atractylodes lancea by Protection of the Intestinal Mucosal Barrier.

Atractylodes lancea (Thunb.) DC. (AL) is used in traditional Chinese medicine for the treatment of spleen-deficiency syndrome (SDS). Bran-processed Atractylodes lancea (BAL) has been found to be more effective than unprocessed AL. However, the compound in BAL active against SDS remains unclear. The pharmacological efficacy of BAL and its mechanism of action against SDS were investigated by HPLC-ELSD. Candidate compound AA (atractyloside A) in AL and BAL extracts was identified by HPLC-MS analysis. AA was tested in a rat model of SDS in which body weight, gastric residual rate, and intestinal propulsion were measured, and motilin (MTL), gastrin (GAS), and c-Kit were quantified by enzyme-linked immunosorbent assay. Potential targets and associated pathways were identified based on network pharmacology analysis. mRNA expression levels were measured by qRT-PCR and protein expression levels were measured by Western blot analysis and immunohistochemistry. AA increased body weight, intestinal propulsion, MTL, GAS, and c-Kit levels, while decreasing gastric residual volume and intestinal tissue damage, as same as Epidermal Growth Factor Receptor and Proliferating Cell Nuclear Antigen levels. Seventy-one potential pharmacologic targets were identified. Analysis of protein interaction, Gene Ontology (GO) functional analysis, pathway enrichment analysis, and docking and molecular interactions highlighted MAPK signaling as the potential signal transduction pathway. Validation experiments indicated that treatment with AA increased MTL, GAS, ZO-1, and OCLN levels, while reducing AQP1, AQP3, and FGF2 levels. In addition, phosphorylation of p38 and myosin light-chain kinase (MLCK) expression were inhibited. AA improved gastrointestinal function by protecting the intestinal mucosal barrier via inhibition of the p38 MAPK pathway. The results have clinical implications for the therapy of SDS.

Deep-Fried Atractylodis Rhizoma Protects against Spleen Deficiency-Induced Diarrhea through Regulating Intestinal Inflammatory Response and Gut Microbiota.

According to the theories of traditional Chinese medicine, spleen deficiency often leads to diarrhea, and deep-fried Atractylodis Rhizoma (DAR) is commonly used for the treatment. However, the association between spleen deficiency and diarrhea remains unclear. The present study aimed to investigate the therapeutic effect of DAR for the treatment of diarrhea caused by spleen deficiency and analyze the related mechanisms. It was found that a high dose group of an ethanolic extract of deep-fried Atractylodis Rhizoma (EEDAR-H) significantly inhibited weight loss, diarrhea, and pathological changes in colon tissue induced by rhubarb. EEDAR-H was found to significantly reduce the level of intestinal inflammatory cytokines and increase the expression of gastrointestinal motility hormones. In addition, EEDAR-H significantly increased the expression of aquaporin 3 (AQP3) and aquaporin 8 (AQP8) and restored abnormal water metabolism; Shen-Ling-Bai-Zhu-San (SLBZS) induced the same effect as EEDAR-H. Additional tests on the mechanism found that EEDAR-H and SLBZS promoted the integrity of the intestinal barrier. Both significantly increased the expression of the tight junction protein ZO-1 and Occludin, inhibited the phosphorylation of p38MAPK and MLC, and significantly reduced the expression levels of PAR-2. Analysis of the gut microbiota indicated that overall changes in its structure were reversed after treatment with EEDAR-H or SLBZS, in addition to significant modulation of the abundance of different phyla. At the genus level, EEDAR-H or SLBZS significantly reduced the levels of potential pathogens and increased those of beneficial bacteria.

[Study on dryness effect of Atractylodis Rhizoma based on theory of dry-dry and dryness-induced Yin deficiency].

In order to establish a more perfect evaluation system for dryness effect of Atractylodis Rhizoma, determine the main dry parts of Atractylodis Rhizoma，and further define the mechanism of stir-baked Atractylodis Rhizoma in reducing the dryness. The healthy rats were given with different doses of water extract and volatile oil of raw Atractylodis Rhizoma and stir-baked Atractylodis Rhizoma for 21 days. Based on the theory of the dry-dry and dryness-induced Yin deficiency, the amount of drinking water, tissue morphology of submandibular glands, urine volume and the expression of aquaporin 2 (AQP2) in the kidneys, as well as blood rheology, ratio of cAMP/cGMP in serum and the content of Na⁺-K⁺-ATP enzyme were selected as the evaluation indexes. The results indicated that the rats with high dose volatile oil from raw Atractylodis Rhizoma had a significant increase in the amount of drinking water, urine volume, blood viscosity, ratio of cAMP/cGMP and content of Na⁺-K⁺-ATP enzyme in the serum（P<0.05）as compared with the soybean oil group; meanwhile, atrophy of submandibular acinar gland was obvious，and the expression of aquaporin 2 was reduced significantly（P<0.05）. There were significant differences between volatile oil high dose group of raw Atractylodis Rhizoma and volatile oil high dose group of stir-baked Atractylodis Rhizoma. There was no significant difference between the water extract groups of raw and stir-baked Atractylodis Rhizoma and the saline group. A comprehensive evaluation system for the dryness effect of Atractylodis Rhizoma was established. It was confirmed that the volatile oil part was the main dry part of Atractylodis Rhizoma. It revealed that the mechanism of dryness effect of Atractylodis Rhizoma was not only related to the decrease of the total content of the volatile oil, but also may be related to the transformation of dryness components in the volatile oil. It provides references for the study of material basis of Atractylodis Rhizoma dryness, provides an experimental basis for the clinical application of Atractylodis Rhizoma, further clarifies the mechanism of stir-baked Atractylodis Rhizoma in reducing the dryness, and provides thoughts for the evaluation of other dry traditional Chinese medicines.

Myosin IIA-related Actomyosin Contractility Mediates Oxidative Stress-induced Neuronal Apoptosis.

Oxidative stress-induced neuronal apoptosis plays an important role in the progression of central nervous system (CNS) diseases. In our study, when neuronal cells were exposed to hydrogen peroxide (H2O2), an exogenous oxidant, cell apoptosis was observed with typical morphological changes including membrane blebbing, neurite retraction and cell contraction. The actomyosin system is considered to be responsible for the morphological changes, but how exactly it regulates oxidative stress-induced neuronal apoptosis and the distinctive functions of different myosin II isoforms remain unclear. We demonstrate that myosin IIA was required for neuronal contraction, while myosin IIB was required for neuronal outgrowth in normal conditions. During H2O2-induced neuronal apoptosis, myosin IIA, rather than IIB, interacted with actin filaments to generate contractile forces that lead to morphological changes. Moreover, myosin IIA knockout using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein-9 nuclease (CRISPR/Cas9) reduced H2O2-induced neuronal apoptosis and the associated morphological changes. We further demonstrate that caspase-3/Rho-associated kinase 1 (ROCK1) dependent phosphorylation of myosin light chain (MLC) was required for the formation of the myosin IIA-actin complex. Meanwhile, either inhibition of myosin II ATPase with blebbistatin or knockdown of myosin IIA with siRNA reversely attenuated caspase-3 activation, suggesting a positive feedback loop during oxidative stress-induced apoptosis. Based on our observation, myosin IIA-actin complex contributes to actomyosin contractility and is associated with the positive feedback loop of caspase-3/ROCK1/MLC pathway. This study unravels the biochemical and mechanistic mechanisms during oxidative stress-induced neuronal apoptosis and may be applicable for the development of therapies for CNS diseases.

YiQiFuMai Powder Injection Protects against Ischemic Stroke via Inhibiting Neuronal Apoptosis and PKCδ/Drp1-Mediated Excessive Mitochondrial Fission.

YiQiFuMai (YQFM) powder injection has been reported to be used in cardiovascular and nervous system diseases with marked efficacy. However, as a treatment against diseases characterized by hypoxia, lassitude, and asthenia, the effects and underlying mechanisms of YQFM in neuronal mitochondrial function and dynamics have not been fully elucidated. Here, we demonstrated that YQFM inhibited mitochondrial apoptosis and activation of dynamin-related protein 1 (Drp1) in cerebral ischemia-injured rats, producing a significant improvement in cerebral infarction and neurological score. YQFM also attenuated oxidative stress-induced mitochondrial dysfunction and apoptosis through increasing ATP level and mitochondria membrane potential (Δψm), inhibiting ROS production, and regulating Bcl-2 family protein levels in primary cultured neurons. Moreover, YQFM inhibited excessive mitochondrial fission, Drp1 phosphorylation, and translocation from cytoplasm to mitochondria induced by oxidative stress. We provided the first evidence that YQFM inhibited the activation, association, and translocation of PKCδ and Drp1 upon oxidative stress. Taken together, we demonstrate that YQFM ameliorates ischemic stroke-induced neuronal apoptosis through inhibiting mitochondrial dysfunction and PKCδ/Drp1-mediated excessive mitochondrial fission. These findings not only put new insights into the unique neuroprotective properties of YQFM associated with the regulation of mitochondrial function but also expand our understanding of the underlying mechanisms of ischemic stroke.

Ginsenoside Rg1 Protects against Oxidative Stress-induced Neuronal Apoptosis through Myosin IIA-actin Related Cytoskeletal Reorganization.

Oxidative stress-induced cytoskeletal dysfunction of neurons has been implicated as a crucial cause of cell apoptosis or death in the central nervous system (CNS) diseases, such as neurodegenerative and psychiatric diseases. The application of neuroprotectants rescuing the neurons from cytoskeletal damage and apoptosis can be a potential treatment for these CNS diseases. Ginsenoside Rg1 (Rg1), one of the major active components of ginseng, has been reported possessing notable neuroprotective activities. However, there is rare report about its effect on cytoskeleton and its undergoing mechanism. The current study is to reveal the regulatory effects of Rg1 on cytoskeletal and morphological lesion in oxidative stress-induced neuronal apoptosis. The results demonstrated that pre-treatment with Rg1 (0.1-10 µM) attenuated hydrogen peroxide (H2O2)-induced neuronal apoptosis and oxidative stress through reducing the intracellular reactive oxygen species (ROS) production and methane dicarboxylic aldehyde (MDA) level. The Rg1 treatment also abolished H2O2-induced morphological changes, including cell rounding, membrane blebbing, neurite retraction and nuclei condensation, which were generated by myosin IIA-actin interaction. These effects were mediated via the down-regulation of caspase-3, ROCK1 (Rho-associated kinase1) activation and myosin light chain (MLC, Ser-19) phosphorylation. Furthermore, inhibiting myosin II activity with blebbistatin partly blocked the neuroprotective effects of Rg1. The computer-aided homology modelling revealed that Rg1 preferentially positioned in the actin binding cleft of myosin IIA and might block the binding of myosin IIA to actin filaments. Accordingly, the neuroprotective mechanism of Rg1 is related to the activity that inhibits myosin IIA-actin interaction and the caspase-3/ROCK1/MLC signaling pathway. These findings put some insights into the unique neuroprotective properties of Rg1 associated with the regulation of myosin IIA-actin cytoskeletal structure under oxidative stress and provide experimental evidence for Rg1 in CNS diseases.

Ruscogenin Attenuates Cerebral Ischemia-Induced Blood-Brain Barrier Dysfunction by Suppressing TXNIP/NLRP3 Inflammasome Activation and the MAPK Pathway.

Ruscogenin, an important steroid sapogenin derived from Ophiopogon japonicus, has been shown to inhibit cerebral ischemic injury. However, its potential molecular action on blood-brain barrier (BBB) dysfunction after stroke remains unclear. This study aimed to investigate the effects of ruscogenin on BBB dysfunction and the underlying mechanisms in middle cerebral artery occlusion/reperfusion (MCAO/R)-injured mice and oxygen-glucose deprivation/reoxygenation (OGD/R)-injured mouse brain microvascular endothelial cells (bEnd.3). The results demonstrated that administration of ruscogenin (10 mg/kg) decreased the brain infarction and edema, improved neurological deficits, increased cerebral brain flow (CBF), ameliorated histopathological damage, reduced evans blue (EB) leakage and upregulated the expression of tight junctions (TJs) in MCAO/R-injured mice. Meanwhile, ruscogenin (0.1-10 µM) treatment increased cell viability and trans-endothelial electrical resistance (TEER) value, decreased sodium fluorescein leakage, and modulated the TJs expression in OGD/R-induced bEnd.3 cells. Moreover, ruscogenin also inhibited the expression of interleukin-1ß (IL-1ß) and caspase-1, and markedly suppressed the expression of Nucleotide-binding domain (NOD)-like receptor family, pyrin domain containing 3 (NLRP3) and thiredoxin-interactive protein (TXNIP) in vivo and in vitro. Furthermore, ruscogenin decreased reactive oxygen species (ROS) generation and inhibited the mitogen-activated protein kinase (MAPK) pathway in OGD/R-induced bEnd.3 cells. Our findings provide some new insights into its potential application for the prevention and treatment of ischemic stroke.

A combination of four effective components derived from Sheng-mai san attenuates hydrogen peroxide-induced injury in PC12 cells through inhibiting Akt and MAPK signaling pathways.

The present study was designed to investigate whether a combination of four effective components derived from Sheng-mai san (SMXZF; ginsenoside Rb1: ginsenoside Rg1: DT-13: Schizandrol A as 6 : 9 : 4 : 5) could attenuate hydrogen peroxide (H2O2)-induced injury in PC12 cells, focusing on the Akt and MAPK pathways . The PC12 cells were exposed to H2O2 (400 µmol·L(-1)) for 1 h in the presence or absence of SMXZF pre-treatment for 24 h. Cell viability was measured by MTT assay. The efflux of lactate dehydrogenase (LDH), the intracellular content of malondialdehyde (MDA), the activities of superoxide dismutase (SOD), and caspase-3 were also determined. Cell apoptosis was measured by Hoechst 33342 staining and Annexin V-FITC/PI staining method. The expression of Bcl-2, Bax, cleaved caspase-3, Akt, and MAPKs were detected by Western blotting analyses. SMXZF pretreatment significantly increased the cell viability and SOD activity and improved the cell morphological changes, while reduced the levels of LDH and MDA at the concentrations of 0.1, 1 and 10 µg·mL(-1). SMXZF also inhibited H2O2-induced apoptosis in PC12 cells. Moreover, SMXZF reduced the activity of caspase-3, up-regulated the protein ratio of Bcl-2 and Bax and inhibited the expression of cleaved caspase-3, p-Akt, p-p38, p-JNK and p-ERK1/2 in H2O2-induced PC12 cells. Co-incubation of Akt inhibitor or p38 inhibitor partly attenuated the protection of SMXZF against H2O2-injured PC12 cells. In conclusion, our findings suggested that SMXZF attenuated H2O2-induced injury in PC12 cells by inhibiting Akt and MAPKs signaling pathways, which might shed insights on its neuroprotective mechanism.

Migration-inducing gene 7 promotes tumorigenesis and angiogenesis and independently predicts poor prognosis of epithelial ovarian cancer.

Epithelial ovarian carcinomas (EOC) cause more mortality than any other cancer of the female reproductive system. New therapeutic approaches to reduce EOC mortality have been largely unsuccessful due to the poor understanding of the mechanisms underlying EOC proliferation and metastasis. Progress in EOC treatment is further hampered by a lack of reliable prognostic biomarkers for early risk assessment. In this study, we identify that Migration-Inducting Gene 7 (MIG-7) is specifically induced in human EOC tissues but not normal ovaries or ovarian cyst. Ovarian MIG-7 expression strongly correlated with EOC progression. Elevated MIG-7 level at the time of primary cytoreductive surgery was a strong and independent predictor of poor survival of EOC patients. Cell and murine xenograft models showed that MIG-7 was required for EOC proliferation and invasion, and MIG-7 enhanced EOC-associated angiogenesis by promoting the expression of vascular endothelial growth factor. Inhibiting MIG-7 by RNA interference in grafted EOC cells retarded tumor growth, angiogenesis and improved host survival, and suppressing MIG-7 expression with a small molecule inhibitor D-39 identified from the medicinal plant Liriope muscari mitigated EOC growth and invasion and specifically abrogated the expression of vascular endothelial growth factor. Our data not only reveal a critical function of MIG-7 in EOC growth and metastasis and support MIG-7 as an independent prognostic biomarker for EOC, but also demonstrate that therapeutic targeting of MIG-7 is likely beneficial in the treatment of EOC.

YiQiFuMai Powder Injection Ameliorates Cerebral Ischemia by Inhibiting Endoplasmic Reticulum Stress-Mediated Neuronal Apoptosis.

YiQiFuMai (YQFM) powder injection as a modern preparation derived from Sheng Mai San, a traditional Chinese medicine, has been widely used in the treatment of cardiovascular and cerebrovascular diseases. However, its neuroprotective effect and underlying mechanism in cerebral ischemia remain to be explored. The present study was designed to investigate the neuroprotective effect of YQFM on endoplasmic reticulum (ER) stress-mediated neuronal apoptosis in the permanent middle cerebral artery occlusion- (MCAO-) injured mice and the oxygen-glucose deprivation- (OGD-) induced pheochromocytoma (PC12) cells. The results showed that single administration of YQFM (1.342 g/kg, i.p.) could reduce the brain infarction and improve the neurological deficits and the cerebral blood flow (CBF) after MCAO for 24 h in mice. Moreover, incubation with YQFM (100, 200, and 400 µg/mL) could increase the cell viability, decrease the caspase-3 activity, and inhibit the cell apoptosis in OGD-induced PC12 cells for 12 h. In addition, YQFM treatment could significantly modulate cleaved caspase-3 and Bcl-2 expressions and inhibit the expressions of ER stress-related marker proteins and signaling pathways in vivo and in vitro. In conclusion, our findings provide the first evidence that YQFM ameliorates cerebral ischemic injury linked with modulating ER stress-related signaling pathways, which provided some new insights for its prevention and treatment of cerebral ischemia diseases.