Identification of SARS-CoV-2 inhibitors using lung and colonic organoids.

There is an urgent need to create novel models using human disease-relevant cells to study severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) biology and to facilitate drug screening. Here, as SARS-CoV-2 primarily infects the respiratory tract, we developed a lung organoid model using human pluripotent stem cells (hPSC-LOs). The hPSC-LOs (particularly alveolar type-II-like cells) are permissive to SARS-CoV-2 infection, and showed robust induction of chemokines following SARS-CoV-2 infection, similar to what is seen in patients with COVID-19. Nearly 25% of these patients also have gastrointestinal manifestations, which are associated with worse COVID-19 outcomes1. We therefore also generated complementary hPSC-derived colonic organoids (hPSC-COs) to explore the response of colonic cells to SARS-CoV-2 infection. We found that multiple colonic cell types, especially enterocytes, express ACE2 and are permissive to SARS-CoV-2 infection. Using hPSC-LOs, we performed a high-throughput screen of drugs approved by the FDA (US Food and Drug Administration) and identified entry inhibitors of SARS-CoV-2, including imatinib, mycophenolic acid and quinacrine dihydrochloride. Treatment at physiologically relevant levels of these drugs significantly inhibited SARS-CoV-2 infection of both hPSC-LOs and hPSC-COs. Together, these data demonstrate that hPSC-LOs and hPSC-COs infected by SARS-CoV-2 can serve as disease models to study SARS-CoV-2 infection and provide a valuable resource for drug screening to identify candidate COVID-19 therapeutics.

Checkpoint kinase 2 controls insulin secretion and glucose homeostasis.

After the discovery of insulin, a century ago, extensive work has been done to unravel the molecular network regulating insulin secretion. Here we performed a chemical screen and identified AZD7762, a compound that potentiates glucose-stimulated insulin secretion (GSIS) of a human ß cell line, healthy and type 2 diabetic (T2D) human islets and primary cynomolgus macaque islets. In vivo studies in diabetic mouse models and cynomolgus macaques demonstrated that AZD7762 enhances GSIS and improves glucose tolerance. Furthermore, genetic manipulation confirmed that ablation of CHEK2 in human ß cells results in increased insulin secretion. Consistently, high-fat-diet-fed Chk2-/- mice show elevated insulin secretion and improved glucose clearance. Finally, untargeted metabolic profiling demonstrated the key role of the CHEK2-PP2A-PLK1-G6PD-PPP pathway in insulin secretion. This study successfully identifies a previously unknown insulin secretion regulating pathway that is conserved across rodents, cynomolgus macaques and human ß cells in both healthy and T2D conditions.

[Quality control mechanism of mitochondria by 3,4-dihydroxybenzaldehyde through OGT-PINK1 pathway].

Based on the O-GlcNAc transferase(OGT)-PTEN-induced putative kinase 1(PINK1) pathway, the mechanism of 3,4-dihydroxybenzaldehyde(DBD) on mitochondrial quality control was investigated. Middle cerebral artery occlusion/reperfusion(MCAO/R) rats were established. SD rats were randomized into sham operation group(sham), model group(MCAO/R), DBD-L group(5 mg·kg~(-1)), and DBD-H group(10 mg·kg~(-1)). After 7 days of administration(ig), MCAO/R was induced in rats except the sham group with the suture method. Twenty-four h after reperfusion, the neurological function and the percentage of cerebral infarct area were measured. Based on hematoxylin and eosin(HE) staining and Nissl staining, the pathological damage of cerebral neurons was examined. Then the ultrastructure of mitochondria was observed under the electron microscope, and the co-localization of light chain-3(LC3), sequestosome-1(SQSTM1/P62), and Beclin1 was further detected by immunofluorescence staining. It has been reported that the quality of mitochondria can be ensured by inducing mitochondrial autophagy through the OGT-PINK1 pathway. Therefore, Western blot was employed to detect the expression of OGT, mitophagy-related proteins PINK1 and E3 ubiquitin ligase(Parkin), and mitochondrial kinetic proteins dynamin-like protein 1(Drp1) and optic atrophy 1(Opa1). The results showed that MCAO/R group had neurological dysfunction, large cerebral infarct area(P<0.01), damaged morphological structure of neurons, decreased number of Nissl bodies, mitochondrial swelling, disappearance of mitochondrial cristae, decrease of cells with LC3 and Beclin1, rise of cells with P62(P<0.01), inhibited expression of OGT, PINK1, and Parkin, up-regulated expression of Drp1, and down-regulated expression of Opa1 compared with the sham group(P<0.01). However, DBD improved the behavioral deficits and mitochondrial health of MCAO/R rats, as manifested by the improved morphology and structure of neurons and mitochondria and the increased Nissl bodies. Moreover, DBD increased cells with LC3 and Beclin1 and decreased cells with P62(P<0.01). In addition, DBD promoted the expression of OGT, PINK1, Parkin, and Opa1 and inhibited the expression of Drp1, enhancing mitophagy(P<0.05, P<0.01). In conclusion, DBD can trigger PINK1/Parkin-mediated brain mitophagy through the OGT-PINK1 pathway, which plays a positive role in maintaining the health of the mitochondrial network. This may be a mitochondrial therapeutic mechanism to promote nerve cell survival and improve cerebral ischemia/reperfusion injury.

Integrative analysis of transcriptomics and metabolomics to reveal the melanogenesis pathway of muscle and related meat characters in Wuliangshan black-boned chickens.

BACKGROUND: Melanin is an important antioxidant in food and has been used in medicine and cosmetology. Chicken meat with high melanin content from black-boned chickens have been considered a high nutritious food with potential medicinal properties. The molecular mechanism of melanogenesis of skeletal muscle in black-boned chickens remain poorly understood. This study investigated the biological gene-metabolite associations regulating the muscle melanogenesis pathways in Wuliangshan black-boned chickens with two normal boned chicken breeds as control. RESULTS: We identified 25 differentially expressed genes and 11 transcription factors in the melanogenesis pathways. High levels of the meat flavor compounds inosine monophosphate, hypoxanthine, lysophospholipid, hydroxyoctadecadienoic acid, and nicotinamide mononucleotide were found in Wuliangshan black-boned chickens. CONCLUSION: Integrative analysis of transcriptomics and metabolomics revealed the dual physiological functions of the PDZK1 gene, involved in pigmentation and/or melanogenesis and regulating the phospholipid signaling processes in muscle of black boned chickens.

Inflammatory Cytokines, Adipocytokines, and Th17/Treg Balance in Patients with Nonalcoholic Fatty Liver Disease following Administration of Dahuang Zhechong Pills.

Objectives: The occurrence and development of nonalcoholic fatty liver disease (NAFLD) is related to lipid peroxidation, imbalance of inflammatory response factors, and immune function disorder. This study was conducted with the purpose of investigating the expression levels of inflammatory cytokines and adipocytokines and Th17/Treg balance in NAFLD patients treated with Dahuang Zhechong pills (DHZCPs). Methods: The study recruited 100 NAFLD patients who were then arranged into the test group and control group. Patients in the test group were treated with DHZCPs, while patients in the control group were untreated. Peripheral TH17 and Treg cells were detected by flow cytometry, and peripheral IL-17, IL-10, hs-CRP, and TNF-α expression levels were determined by enzyme-linked immunosorbent assay (ELISA) methods. The concentrations of ghrelin, leptin, and adiponectin were quantitatively examined. Results: The levels of TC, TG, ALT, and AST were declined but the level of HDL-C was increased in NAFLD patients treated with DHZCPs compared with untreated patients (P < 0.05). The ratio of Th17/Treg in NAFLD patients treated with DHZCPs was (1.52 ± 0.21), which was significantly lower than (2.39 ± 0.45) of untreated patients (P < 0.05). The levels of IL-17, hs-CRP, and TNF-α were lower, but the level of IL-10 was higher in NAFLD patients treated with DHZCPs than that in untreated patients (P < 0.05). The expression levels of ghrelin and adiponectin in NAFLD patients treated with DHZCPs were evidently higher than those in untreated patients (P < 0.01), and the expression level of leptin in NAFLD patients treated with DHZCPs was evidently lower than that in untreated patients (P < 0.01). Conclusions: Administration of DHZCPs regulates the immune function of NAFLD patients by keeping Th17/Treg balance and affecting the levels of inflammatory cytokines and adipocytokines.

[Effect of ethanol extract of Gastrodiae Rhizoma on mitochondrial dysfunction in cerebral ischemia-reperfusion injury].

This study observed the effects of ethanol extract of Gastrodiae Rhizoma(GE) on multiple aspects of mitochondrial dysfunction by investigating the mitochondria isolated from rat brains subjected to focal middle cerebral artery occlusion/reperfusion(MCAO/R). SD rats were randomly divided into a sham operation group(Sham), a model group(MCAO/R), low-and high-dose ethanol extract of GE groups(262.3 and 524.6 mg·kg~(-1)), and a nimodipine group(Nim, 15 mg·kg~(-1)). After continuous intragastric administration for 7 days, the MCAO/R model was induced in rats by the suture method. The neurological function and percentage of cerebral infarction volume were measured 24 h after reperfusion, and mitochondrial ultrastructure was observed under an electron microscope. Mitochondria were separated by gradient centrifugation and detected for reactive oxygen species(ROS), malondialdehyde(MDA), respiratory chain enzyme complex Ⅰ-Ⅳ activity, mitochondrial permeability transition pore(mPTP), mitochondrial membrane potential(MMP), and mitochondrial adenosine triphosphate(ATP) content. Enzyme-linked immunosorbent assay(ELISA) was used to detect the expression of cytochrome C(Cyt C) in different sites. TUNEL staining was used to observe the apoptosis of brain tissues in each group, and Western blot was used to detect the expression of B-cell lymphoma 2(Bcl-2) and Bcl-2-associated X protein(Bax) in brain tissues. The experimental results revealed that compared with the Sham group, the MCAO/R group showed evident neurological dysfunction and cerebral infarction(P<0.01) accompanied by mitochondrial swelling and crest disappearance, increased ROS level and MDA content, inhibited activity of respiratory chain enzyme complex Ⅰ-Ⅳ, abnormal opening of mPTP, and reduced MMP and mitochondrial ATP(P<0.01). Besides, many Cyt C was released from mitochondria into the cytoplasm to induce apoptosis(P<0.01). The ethanol extract of GE positively affected the behavior deficit and mitochondrial health of MCAO/R rats, with the manifestations of decreased production of ROS and MDA(P<0.01), potentiated activity of mitochondrial respiratory chain enzyme complex Ⅰ-Ⅳ, and restored the level of mPTP(P<0.05). In addition, the ethanol extract of GE reduced the loss of MMP and the escape of Cyt C to the cytoplasm(P<0.05), reduced the number of TUNEL positive cells(P<0.01) accompanied by the decrease in Bax and the up-regulation of Bcl-2(P<0.01), and increased the level of ATP(P<0.01). In conclusion, GE ethanol extract has a protective effect against MCAO/R-induced mitochondrial dysfunction, and the mechanism may be related to the regulation of oxidative stress, maintenance of functional morphology of mitochondria, and inhibition of endogenous apoptosis.

[Effect of Desmodium renifolium on ovariectomized rat model of osteoporosis via BMP-2/Smads signaling pathway].

This study aimed to investigate the effect of Desmodium renifolium(Linn.) Schindl on ovariectomized rat model of osteoporosis and explore the underlying mechanism. Rats were randomized into a sham group, a model group, a Xianlin Gubao group, and low-, medium-, and high-dose D. renifolium groups. Bilateral oophorectomy was performed in other groups except sham group(removal of bilateral peri-ovarian adipose tissue). The sham group and model group were administrated with equal volume of 0.5% CMC-Na, Xianlin Gubao group with Xianlin Gubao, and D. renifolium groups with different concentrations of alcoholic extract of D. renifolium once a day for 14 weeks. The body weight of rats were recorded during the experiment. The levels of estradiol(E_2), 1,25-dihydroxyvitamin D_3 [1,25(OH)_2D_3], calcium(Ca), and phosphorus(P) in serum were determined after the administration. The femur microstructure was analyzed via micro-CT. RT-PCR and Western blot were employed to respectively determine the mRNA and protein levels of bone morphogenetic protein 2(BMP-2), Runt-related transcription factor 2(Runx2), and osterix(OSX) in the tibia of rats. The results indicated that D. renifolium significantly inhibited the weight growth, improved the uterus appearance, elevated the levels of E_2, Ca, P, and 1,25(OH)_2D_3 in serum, increased the number and reduced the fracture of bone trabeculae, ameliorated the bone microstructure parameters, and up-regulated the mRNA and protein levels of BMP-2, Runx2, and OSX. All the results demonstrated that D. renifolium had significant protective effect on the ovariectomized rat model of osteoporosis by regulating the BMP-2/Smads signaling pathway.

Parkin overexpression reduces inflammation-mediated cardiomyocyte apoptosis through activating Nrf2/ARE signaling pathway.

Inflammation has been acknowledged as one of the pathological alterations in various cardiovascular disorders. Parkin has been found to be associated with mitochondrial protection. In the present study, we explored the influence of Parkin overexpression on cardiomyocyte induced by LPS-mediated inflammation response. Our results demonstrated that cardiomyocyte viability was reduced and apoptotic rate was increased upon LPS treatment, an effect that may be caused by cardiomyocyte oxidative stress. At the molecular levels, LPS treatment promoted ROS production, a result that was followed by a drop in the levels of anti-oxidants. Interestingly, Parkin overexpression significantly promoted cardiomyocyte survival and this cardioprotective was attributable to the anti-oxidative property. Parkin overexpression enhanced the expression of anti-oxidative factors such as GSH, SOD and GPX, resulting into depressed ROS production. Further, we found that Parkin modulated cellular anti-oxidative capacity through the Nrf2/ARE signaling pathway. This finding demonstrates that oxidative stress could be considered as the core of inflammation response. Further, therapeutic approaches targeting Parkin would improve cardiomyocyte anti-oxidative capacity through activating Nrf2/ARE signaling pathway.

Epigenetic silencing of microRNA-204 by Helicobacter pylori augments the NF-κB signaling pathway in gastric cancer development and progression.

Helicobacter pylori infection induces gastric cancer (GC) development through a progressive cascade; however, the roles of the microRNAs that are involved in the cascade and the underlying mechanisms are still unclear. Here, we found that microRNA-204 was suppressed in gastric mucosal cells in response to H.pylori infection and downregulated in GC tissues due to aberrant methylation of the promoter of its host gene, TRPM3. Helicobacter pylori induced a progressive downregulation of microRNA-204 from superficial gastritis to intestinal metaplasia, with an accompanying increment of the methylated levels of CpG sites in the TRPM3 promoter. With the GC cellular models of AGS, MGC-803 or BGC-823, we found that microRNA-204 suppressed the tumor necrosis factor (TNF)-α-induced activation of NF-κB signaling pathways and, in animal models, inhibited tumor growth and metastasis. The conditional supernatant of microRNA-204 overexpression GC cells led to reduced tube formation of human umbilical vein endothelial cells. A target gene for microRNA-204 was BIRC2, and in GC cells, BIRC2 knockdown recapitulated the biological phenotype of microRNA-204 overexpression. BIRC2 overexpression promoted the metastasis of GC cells and rescued the inhibition activities of microRNA-204 on cell migration and the NF-κB signaling pathway. Moreover, lower microRNA-204 and higher BIRC2 expression levels were associated with a poorer prognosis of GC patients. These results demonstrate that epigenetic silencing of microRNA-204 induced by H.pylori infection augments the NF-κB signaling pathway in H.pylori-induced gastritis and GC, potentially providing novel intervention targets for these diseases. MicroRNA-204 was epigenetically down-regulated by H. pylori infection in gastric mucosal cells. It led to enhanced BIRC2 expression level and BIRC2/TNF-a/NF-kB signaling pathway activities, which promoted angiogenesis and metastasis of gastric cancer cells.

Antidepressant-like mechanism of honokiol in a rodent model of corticosterone-induced depression.

Depression is closely linked to hypothalamus-pituitary-adrenal axis hyperactivity. Honokiol, a biphenolic lignan compound obtained from the traditional Chinese medicine Magnolia officinalis, can reduce the activity of the hypothalamus-pituitary-adrenal axis and improve depression-like behavior caused by hypothalamus-pituitary-adrenal axis hyperactivity. The current study investigated the specific mechanism of action of this effect. A depression model was established by repeated injections of corticosterone to study the antidepressant-like effect of honokiol and its potential mechanism. Honokiol prevented the elevated activity of the hypothalamus-pituitary-adrenal axis and the depression-like behavior induced by corticosterone. Treatment with honokiol resulted in greater glucocorticoid receptor mRNA expression, greater glucocorticoid receptor-positive expression, and a greater ratio of glucocorticoid receptor to the mineralocorticoid receptor in the hippocampus. Moreover, honokiol treatment led to lower levels of interleukin-1ß in serum and the positive expression of the interleukin-1ß receptor in the hippocampus. These results demonstrate that the antidepressant-like mechanism of honokiol, which has effects on inflammatory factors, may act through restoring the typical activity of the hypothalamus-pituitary-adrenal axis by regulating the glucocorticoid receptor-mediated negative feedback mechanism and the balance between glucocorticoid and mineralocorticoid receptors.

Protective Effect of 4-Methoxy Benzyl Alcohol on the Blood-Brain Barrier after Cerebral Ischemia Reperfusion Injury.

Damage of the blood-brain barrier (BBB) during the process of cerebral ischemic injury is a key factor that influences the therapeutic efficacy to the cerebral ischemic injury. This work was designed to investigate the mechanisms underlying the protective effects of 4-methoxy benzyl alcohol (4-MA) on the BBB by developing a cerebral ischemia/reperfusion model of rats (MCAO/R). The MCAO/R was developed through a thread embolism method. The neurologic scales, the brain infarct rate, and the Evans blue (EB) contents of the brains were detected. Meanwhile, the release of nitric oxide (NO) and activities of NO synthase (NOS) in brain tissues were measured. Western blotting analyses were also used to assess the protein expressions of aquaporin-4 (AQP-4), occludin, and claudin-5 in brain tissue. After rats were pretreated with different concentrations of 4-MA, the neurologic scores, the infarct rate, and the EB contents in the brain tissues were significantly decreased. The release of NO and the activities of neuronal NOS and inducible NOS were notably inhibited. Furthermore, the protein expression of AQP-4 was markedly decreased, whereas the protein expressions of claudin-5 and occludin were significantly increased. In conclusion, the 4-MA decreases the permeability of BBB when focal cerebral ischemia occurs. The inhibition of the NOS pathways, the attenuation of the protein expression of AQP-4, and the enhancement of the expressions of the tight junction proteins might contribute to the protective effects of 4-MA on the BBB.

Systematic network assessment of the carcinogenic activities of cadmium.

Cadmium has been defined as type I carcinogen for humans, but the underlying mechanisms of its carcinogenic activity and its influence on protein-protein interactions in cells are not fully elucidated. The aim of the current study was to evaluate, systematically, the carcinogenic activity of cadmium with systems biology approaches. From a literature search of 209 studies that performed with cellular models, 208 proteins influenced by cadmium exposure were identified. All of these were assessed by Western blotting and were recognized as key nodes in network analyses. The protein-protein functional interaction networks were constructed with NetBox software and visualized with Cytoscape software. These cadmium-rewired genes were used to construct a scale-free, highly connected biological protein interaction network with 850 nodes and 8770 edges. Of the network, nine key modules were identified and 60 key signaling pathways, including the estrogen, RAS, PI3K-Akt, NF-κB, HIF-1α, Jak-STAT, and TGF-ß signaling pathways, were significantly enriched. With breast cancer, colorectal and prostate cancer cellular models, we validated the key node genes in the network that had been previously reported or inferred form the network by Western blotting methods, including STAT3, JNK, p38, SMAD2/3, P65, AKT1, and HIF-1α. These results suggested the established network was robust and provided a systematic view of the carcinogenic activities of cadmium in human.

Chinese medicine CGA formula ameliorates DMN-induced liver fibrosis in rats via inhibiting MMP2/9, TIMP1/2 and the TGF-ß/Smad signaling pathways.

AIM: Chinese medicine CGA formula consists of polysaccharide from Cordyceps sinensis mycelia (CS-PS), gypenosides and amygdalin, which is derived from Fuzheng Huayu (FZHY) capsule for treating liver fibrosis. In this study we attempted to confirm the therapeutic effects of CGA formula in dimethylnitrosamine (DMN)-induced liver fibrosis in rats, and to identify the mechanisms of anti-fibrotic actions. METHODS: Rats were injected with DMN (10 mg·kg(-1)·d(-1), ip) for 3 consecutive days per week over a 4-week period. The rats then were orally administered with CGA formula (CS-PS 60 mg·kg(-1)·d(-1), gypenosides 50 mg·kg(-1)·d(-1) and amygdalin 80 mg·kg(-1)·d(-1)) daily in the next 2 weeks. CS-PS, gypenosides or amygdalin alone were administered as individual component controls, whereas colchicine and FZHY were used as positive controls. Serum biomarkers were measured. Hepatic injury, collagen deposition and stellate cell activation were examined. The MMP activities, expression of TIMP protein and proteins involved in the TGF-ß1/Smad signaling pathways in liver tissues were assayed. RESULTS: In DMN-treated rats, administration of CGA formula significantly decreased serum ALT, AST and total bilirubin and hepatic hydroxyproline levels, increased serum albumin level, and attenuated liver fibrosis as shown by histological examination. Furthermore, these effects were comparable to those caused by administration of FZHY, and superior to those caused by administration of colchicine or the individual components of CGA formula. Moreover, administration of CGA formula significantly decreased the protein levels of α-SMA, TGF-ß1, TGF-ß1 receptor (TßR-I), p-TßR-I, p-TßR-II, p-Smad2, p-Smad3, TIMP1 and TIMP2, as well as MMP2 and MMP9 activities in liver tissues of DMN-treated rats. CONCLUSION: Chinese medicine CGA formula ameliorates DMN-induced liver fibrosis in rats, and this effect was likely associated with the down-regulation of MMP2/9 activities, TIMP1/2 protein expression and the TGF-ß1/Smad signaling pathways in the liver.

Pharmacokinetics and effects of demographic factors on blood 25(OH)D3 levels after a single orally administered high dose of vitamin D3.

AIM: To examine the biological consequences and demographic factors that might affect the pharmacokinetics of vitamin D3 after a single high dose intervention in a young Chinese population with vitamin D insufficiency status. METHODS: A total of 28 young subjects (25 to 35 years old) with vitamin D insufficiency status [serum 25(OH)D <30 ng/mL] was recruited in Shanghai, China. The subjects were orally administered a single high dose of vitamin D3 (300 000 IU). Baseline characteristics and blood samples were collected at d 0, 1, 2, 3, 7, 28, 56, 84 and 112 after the intervention. The blood biomarker levels were determined with standardized methods. RESULTS: The intervention markedly increased the blood 25(OH)D3 levels within the first five days (mean Tmax=5.1±2.1 d) and sustained an optimal circulating level of 25(OH)D3 (≥30 ng/mL) for 56 d. After the intervention, body weight and baseline 25(OH)D3 levels were significantly correlated with circulating 25(OH)D3 levels. No adverse events and no consistently significant changes in serum calcium, creatinine, glucose, parathyroid hormone, vitamin D binding protein, or the urinary calcium/reatinine ratio were observed. However, there was a significant increase in phosphorus after the vitamin D3 intervention. Total cholesterol and triglyceride levels were decreased at the end of the trial. CONCLUSION: The pharmacokinetics of vitamin D after intervention were influenced by baseline 25(OH)D3 levels and the body weight of the subjects. The results suggest that a single high oral vitamin D3 intervention is safe and efficient for improving the vitamin D status of young Chinese people with vitamin D insufficiency.

Neuroprotective effect of ethyl acetate extract from gastrodia elata against transient focal cerebral ischemia in rats induced by middle cerebral artery occlusion.

OBJECTIVE: To investigate the protective effect of neuroprotection against transient focal cerebral ischemia of the extract from Tianma (Rhizoma Gastrodiae) and the possible mechanisms underlying the action. METHODS: Cerebral ischemia-reperfusion injury was induced through middle cerebral artery occlusion (MCAO). Adult male Sprague-Dawley rats were randomly divided into four groups: sham-operated, ischemia-reperfusion model, 102.6 mg/kg extract treated and 11.4 mg/kg extract treated groups. The extract was prepared from gastrodia elata with ethyl acetate. The effect of the extract tested on rat neurological deficits and Cerebral index, cerebral infarct volume, brain injury, terminal dexynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and B-cell lymphoma-2 (Bcl-2) positive cells. RESULTS: The extract was able to reduce neurological scores, cerebral index and cerebral infarction rate. The brain injury was also relieved by the extract. The results of immunofluorescence staining analysis indicated that the extract increased the expression of Bcl-2 and reduced TUNEL-positive cells significantly in the extract treated groups. CONCLUSION: These results suggested that the extract relieved ischemic injury induced by transient focal cerebral ischemia in rats, and this neuroprotective effect might be partially due to the attenuated apoptosis pathway.

P-hydroxybenzaldehyde protects Caenorhabditis elegans from oxidative stress and ß-amyloid toxicity.

Introduction: Gastrodia elata is the dried tuber of the orchid Gastrodia elata Bl. It is considered a food consisting of a source of precious medicinal herbs, whose chemical composition is relatively rich. Gastrodia elata and its extracted fractions have been shown to have neuroprotective effects. P-hydroxybenzaldehyde (p-HBA), as one of the main active components of Gastrodia elata, has anti-inflammatory, antioxidative stress, and cerebral protective effects, which has potential for the treatment of Alzheimer's disease (AD). The aim of this study was to verify the role of p-HBA in AD treatment and to investigate its mechanism of action in depth based using the Caenorhabditis elegans (C. elegans) model. Methods: In this study, we used paralysis, lifespan, behavioral and antistress experiments to investigate the effects of p-HBA on AD and aging. Furthermore, we performed reactive oxygen species (ROS) assay, thioflavin S staining, RNA-seq analysis, qPCR validation, PCR Array, and GFP reporter gene worm experiment to determine the anti-AD effects of p-HBA, as well as in-depth studies on its mechanisms. Results: p-HBA was able to delay paralysis, improve mobility and resistance to stress, and delay aging in the AD nematode model. Further mechanistic studies showed that ROS and lipofuscin levels, Aß aggregation, and toxicity were reduced after p-HBA treatment, suggesting that p-HBA ameliorated Aß-induced toxicity by enhancing antioxidant and anti-aging activity and inhibiting Aß aggregation. p-HBA had a therapeutic effect on AD by improving stress resistance, as indicated by the down-regulation of NLP-29 and UCR-11 expression and up-regulation of PQN-75 and LYS-3 expression. In addition, the gene microarray showed that p-HBA treatment played a positive role in genes related to AD, anti-aging, ribosomal protein pathway, and glucose metabolism, which were collectively involved in the anti-AD mechanism of p-HBA. Finally, we also found that p-HBA promoted nuclear localization of DAF-16 and increased the expression of SKN-1, SOD-3, and GST-4, which contributed significantly to inhibition of Aß toxicity and enhancement of antioxidative stress. Conclusion: Our work suggests that p-HBA has some antioxidant and anti-aging activities. It may be a viable candidate for the treatment and prevention of Alzheimer's disease.

4,4'-methylenediphenol reduces Aß-induced toxicity in a Caenorhabditis elegans model of Alzheimer's disease.

Introduction: Gastrodia elata Blume is a widely used medicinal and edible herb with a rich chemical composition. Moreover, prescriptions containing Gastrodia elata are commonly used for the prevention and treatment of cardiovascular, cerebrovascular, and aging-related diseases. Recent pharmacological studies have confirmed the antioxidant and neuroprotective effects of Gastrodia elata, and, in recent years, this herb has also been used in the treatment of Alzheimer's disease (AD) and other neurodegenerative disorders. We have previously shown that 4,4'-methylenediphenol, a key active ingredient of Gastrodia elata, can mitigate amyloid-ß (Aß)-induced paralysis in AD model worms as well as prolong the lifespan of the animals, thus displaying potential as a treatment of AD. Methods: We investigated the effects of 4,4'-methylenediphenol on AD and aging through paralysis, lifespan, and behavioral assays. In addition, we determined the anti-AD effects of 4,4'-methylenediphenol by reactive oxygen species (ROS) assay, lipofuscin analysis, thioflavin S staining, metabolomics analysis, GFP reporter gene worm assay, and RNA interference assay and conducted in-depth studies on its mechanism of action. Results: 4,4'-Methylenediphenol not only delayed paralysis onset and senescence in the AD model worms but also enhanced their motility and stress tolerance. Meanwhile, 4,4'-methylenediphenol treatment also reduced the contents of reactive oxygen species (ROS) and lipofuscin, and decreased Aß protein deposition in the worms. Broad-spectrum targeted metabolomic analysis showed that 4,4'-methylenediphenol administration had a positive effect on the metabolite profile of the worms. In addition, 4,4'-methylenediphenol promoted the nuclear translocation of DAF-16 and upregulated the expression of SKN-1, SOD-3, and GST-4 in the respective GFP reporter lines, accompanied by an enhancement of antioxidant activity and a reduction in Aß toxicity; importantly, our results suggested that these effects of 4,4'-methylenediphenol were mediated, at least partly, via the activation of DAF-16. Conclusion: We have demonstrated that 4,4'-methylenediphenol can reduce Aß-induced toxicity in AD model worms, suggesting that it has potential for development as an anti-AD drug. Our findings provide ideas and references for further research into the anti-AD effects of Gastrodia elata and its active ingredients.

Alcohol extract of Rubia yunnanensis: Metabolic alterations and preventive effects against OGD/R­induced oxidative damage in HT22 cells.

The present study investigated the inhibitory and neuroprotective effects of Rubia yunnanensis alcohol extract (RY-A) on oxidative stress induced by oxygen-glucose deprivation/reoxygenation (OGD/R) in HT22 cells. In vitro cultured HT22 cells were randomly divided into control, OGD/R, OGD/R + 100 µmol/l edaravone and OGD/R + 10, 20 and 40 µg/ml RY-A groups. Oxygen-sugar deprivation was performed with 10 mmol/l sodium dithionite combined with sugar-free DMEM medium for 2 h, followed by re-glycolization and reoxygenation for 2 h to establish an in vitro OGD/R model. Cell morphology was observed under a phase contrast microscope. Cell survival rate was detected by thiazolyl blue and lactate dehydrogenase and oxidative stress-related indexes were detected by commercial kits. The effects and metabolic alterations of RY-A treatment after OGD/R were evaluated using ultra-high performance liquid chromatography and mass spectrometry. Protein levels were further examined by western blotting. The results showed that cells in the OGD/R group were swollen and lacked protrusions, had significantly reduced viability and had significantly elevated oxidative stress-related indexes of reactive oxygen species, nitric oxide levels and malondialdehyde content and significantly reduced activities of the antioxidant enzymes superoxide dismutase and glutathione peroxidase, compared with controls. Compared with the OGD/R group, the RY-A group had significantly improved cell morphology and significantly increased cell viability and in terms of oxidative stress, exhibited significantly reduced reactive oxygen species, nitric oxide levels and malondialdehyde content, as well as significantly increased superoxide dismutase and glutathione peroxidase activities. Metabolomic analysis identified changes in 20 metabolites, including L-tryptophan, ornithine, eicosapentaenoic acid-d5, isosafrole and xanthine. Metabolomics analysis showed that the pathways affected included those related to phenylalanine, tyrosine and tryptophan biosynthesis, the prolactin signaling pathway and amphetamine addiction. These results suggested that RY-A had significant preventive effects on an in vitro model of cerebral ischemia-reperfusion injury simulated by OGD/R and the mechanism may be related to increased tryptophan content, activation of indoleamine 2,3-dioxygenase enzymes and inhibition of oxidative stress.

Ethanol extract of Rubia yunnanensis inhibits carotid atherosclerosis via the PI3K/AKT signaling pathway.

Atherosclerosis is a multifactorial vascular disease caused by endothelial dysfunction. Because of adverse reactions to drugs used to treat atherosclerosis. For example, statins, which significantly reduce the burden of atherosclerotic disease, have been associated with muscle toxicity. There is a need to identify novel drugs for the prevention and treatment of atherosclerosis Rubia yunnanensis is a herbs commonly used in Asian countries for its protective effects against cardiovascular diseases. However, the mechanism of action of R. yunnanensis extract in carotid artery atherosclerosis has not been found. The carotid artery is usually used as a site for clinical evaluation of atherosclerosis. The present study aimed to determine the mechanism of action of R. yunnanensis extract in the inhibition of carotid atherosclerosis in apolipoprotein E gene knockout (ApoE-/-) mice. The mechanism of atherosclerosis inhibition was elucidated by detecting the blood lipid level, carotid artery pathology, and the protein expression of PI3K and AKT. The present study demonstrated that ethanol extract of R. yunnanensis reduced lipid levels, intima damage and carotid lipid accumulation and increased p-PI3K/PI3K and p-AKT/AKT protein levels in ApoE-/- mice fed high-fat diet for 12 weeks. It was hypothesized that the effects of R. yunnanensis extract may be achieved by regulation of the phosphatidylinositol-3-kinase/protein kinase B signaling pathway. Ethanol extract of R. yunnanensis decreased carotid atherosclerosis in ApoE-/- mice fed a high-fat diet via the phosphatidylinositol-3-kinase/protein kinase B signaling pathway. Therefore, R. yunnanensis may be a promising option for treating atherosclerosis in the future.