Integrated UHPLC-QE/MS, transcriptomics and network pharmacology reveal the mechanisms via which Liang-Yan-Yi-Zhen-San promotes the browning of white adipose tissue.

We have previously shown that Liang-Yan-Yi-Zhen-San (LYYZS), an ancient Chinese herbal formula, can promote the browning of white adipose tissue. In this study, we sought to determine which active ingredients of LYYZS mediated its effects on the browning of white adipose tissue. Employing ultra-high performance liquid chromatography-Q-Exactive HF mass spectrometry, a total of 52 LYYZS ingredients were identified. On this basis, 1,560 ingredient-related targets of LYYZS were screened using the HERB databases. Meanwhile, RNA sequencing analysis of the inguinal white adipose tissue of mice produced a total of 3148 genes that were significantly differentially expressed following LYYZS treatment and differentially expressed genes regarded as browning-related targets. Through the network pharmacological analysis, a total of 136 intersection targets were obtained and an ingredient-target-pathway network was established. According to network pharmacology analysis, 10 ingredients containing trans-cinnamaldehyde, genistein, daidzein, calycosin, arginine, coumarin, oleic acid, isoleucine, palmitic acid and tyrosine were regarded as active ingredients of browning of white adipose tissue. Integrated evaluation using chemical analysis, transcriptomics and network pharmacology provides an efficient strategy for discovering the active ingredients involved in how LYYZS promotes the browning of white adipose tissue.

Exploration of the Components and Pharmacological Mechanisms of Keyin Pill-Induced Liver Injury Based on Network Pharmacology.

Background: Keyin pill (KP), a patented medicine in China, is used to treat psoriasis. However, KP has been reported to have liver toxicity, but its toxic substance basis and underlying mechanisms remain unclear. Therefore, this study aimed to explore the pharmacological mechanisms and components of KP-induced liver injury through animal experiments, UPLC-QTOF/MS combined with network pharmacology. Methods: Firstly, based on the immune stress mouse model, liver function parameters and hematoxylin-eosin (H&E) staining were detected to investigate KP-induced liver injury. The UPLC-QTOF/MS method was used to identify the components of KP. CTD database and literature mining were further applied to screen nonliver protective components. Subsequently, the nonliver protective components and their corresponding targets and targets of hepatotoxicity were analyzed by the method of network pharmacology. Finally, key targets from networked pharmacology were examined by the enzyme-linked immunosorbent assay (ELISA) and molecular docking. Results: Our results indicated that KP had hepatotoxicity in male Kunming mice, which could favor hepatocyte necrosis and infiltration of inflammatory cells. A total of 70 nonliver protective compounds were identified and screened. The results of network pharmacology illustrated that methoxsalen, obacunone, limonin, and dictamnine might be the main compounds that caused liver damage. The potential hepatotoxicity mechanisms of KP might be through the IL17 and apoptosis pathways to regulate IL6, TNFα, CASP3, and CASP8 targets, thereby causing inflammation, excessive release of factors, and hepatocyte necrosis. The results of the ELISA experiments indicated that KP could increase the release of IL6 and TNFα inflammatory factors in liver tissues. Molecular docking suggested that methoxsalen, obacunone, limonin, and dictamnine had moderate binding ability with CASP3 and CASP8. Conclusion: In this study, the material basis and potential pharmacological mechanisms of KP-induced liver injury were preliminarily explored. Our research provides the initial theoretical basis for reducing the toxicity of KP.

Atractylenolide III from Atractylodes macrocephala Koidz promotes the activation of brown and white adipose tissue through SIRT1/PGC-1α signaling pathway.

BACKGROUND: Hypothermia is a complex pathophysiological response that can be life-threatening in low-temperature environment because of impaired thermoregulation. However, there is currently no clinically effective drugs that can prevent or treat this disease. Brown adipose tissue (BAT) activation or browning of white adipose tissue (WAT) is a promising therapeutic strategy to prevent or treat hypothermia. Atractylodes macrocephala Koidz extract (AE) and its active compound Atractylenolide III (AIII) has been reported to regulate glycolipid metabolism, which might be relevant to BAT activation. However, the thermogenic effect and mechanism of AE and AIII on adipose tissues have not been explored yet. Therefore, this study firstly investigated the role of AE and AIII on hypothermia by promoting heat production of BAT and WAT. PURPOSE: To explore the anti-cold effect of AE and AIII in cold exposure model and explore their biological function and mechanism underlying thermogenesis. METHODS: The effect of thermogenesis and anti-hypothermia of AE and AIII on C57BL/6J mice were evaluated with several experiment in cold environment, such as toxicity test, cold exposure test, metabolism estimation, histology and immunohistochemistry, and protein expression. Additionally, BAT, inguinal WAT (iWAT) and brown adipocytes were utilized to explore the mechanism of AE and AIII on thermogenesis in vivo and in vitro. Finally, SIRT1 agonist and inhibitor in brown adipocytes to verify that AIII activated BAT through SIRT1/PGC-1α pathway. RESULTS: Both AE and AⅢ could significantly maintain the core body temperature and body surface temperature of mice during cold exposure. Besides, AE and AⅢ could significantly improve the capacity of total antioxidant and glucose, lipid metabolism of mice. In addition, AE and AIII reduced mitochondrial membrane potential and ATP content both in BAT and brown adipocytes, and decreased the size of lipid droplets. Moreover, AE and AⅢ promoted the expression of proteins related to heat production in BAT and iWAT. And AIII might activate BAT via SIRT1/PGC-1α pathway. CONCLUSION: AE and AⅢ were potential candidate drugs that treated hypothermia by improving the heat production capacity of the mice. Mechanistically, they may activate SIRT1/PGC-1α pathway, thus enhancing the function of BAT, and promoting the browning of iWAT, to act as anti-hypothermia candidate medicine.

An integrated multi-omics approach revealed the regulation of melatonin on age-dependent mitochondrial function impair and lipid dyshomeostasis in mice hippocampus.

Melatonin can improve mitochondrial dysfunction associated with the aging process by removing active oxygen, as well as inhibiting lipid peroxidation to maintain biofilm fluidity and resist free radical attack. However, there is poor understanding of the effect of melatonin on age-dependent mitochondrial function and lipid profile changes in brain. In this study, we investigated the energy metabolism of the whole body and brain of mice at 9 months, 13 months, and 25 months of continuous gastric administration of 3 mg/kg/d melatonin once per day morning for two months. In addition, we performed transcriptomic, proteomic and lipidomic analysis in the hippocampus of mice at different ages. Proteomics showed that melatonin regulated mitochondrial electron transport and leucine degradation in mouse hippocampus. Lipomics suggested that the long-chain unsaturated glycerol phospholipids in mouse hippocampus increased in an age-dependent manner, while ceramide and glycerol phospholipids decreased significantly in hippocampus of mouse chronically exposed to melatonin. The combined analysis of proteome and liposome demonstrated that Mpst, Ccsap, Hdhd5, Rpl5 and Flna were the key proteins of the network which involved in the regulation of numerous lipids. Furthermore, ultrastructure observation results illustrated that melatonin could improve the damaged mitochondrial and morphologies of 25-month-old mice hippocampus. In conclusion, we describe a mechanism that age-dependent up-regulation of long-chain unsaturated lipids is a driving risk factor for mitochondrial damage and this effect could be reversed by chronic supplement of low-dose melatonin.

To Explore the Effects of Acupuncture and Medical Treatment at Different Times on the Gastrointestinal Reaction and White Blood Cell Count of Patients with Lung Cancer Chemotherapy.

Objective: To explore and analyze the effects of acupuncture and medical treatment at different times on the gastrointestinal reaction and leukocyte count of patients with lung cancer undergoing chemotherapy. Methods: Select 224 lung cancer chemotherapy patients admitted to our hospital and randomly divide them into three groups: control group (n = 76), study 1 group (n = 78), and study 2 group (n = 70). The control group was treated with tropisetron hydrochloride for 30 minutes before chemotherapy. Study 1 group was given tropisetron hydrochloride and acupuncture combination therapy 30 minutes before chemotherapy. Study 2 group was given tropisetron hydrochloride treatment 30 min before chemotherapy and acupuncture treatment 30 min after chemotherapy. Collect patients' general information and compare the three groups of white blood cell count, G-CSF, GM-CSF levels, quality of life and KPS score, platelets, neutrophils, hemoglobin levels, TCM symptom scores, and the degree of digestive tract reaction. Results: The data of the control group and study groups 1 and 2 are comparable in gender, age, pathological type, etc. (P > 0.05). Before treatment, the white blood cell counts of the three groups were not significantly different (P > 0.05), significantly reduced after treatment, but the difference between the groups was not statistically significant (P > 0.05). The levels of G-CSF and GM-CSF in the three groups were not substantially different before treatment (P > 0.05). The levels of G-CSF and GM-CSF were considerably lowered in all three groups, although the drop in the study group was more significant (P > 0.05) when compared to that in the control group. Before treatment, the quality of life and KPS score of the three groups were not statistically different (P > 0.05). The three groups' quality of life and KPS scores fell after treatment, with the study 1 group experiencing the greatest reduction, followed by the study 2 group and the control group. The levels of platelets, neutrophils, and hemoglobin in all three groups declined dramatically, with the most noticeable reduction in the control group, followed by study 2 and study 1. The difference between the three groups was statistically significant (P < 0.05). The TCM symptom scores of the three groups showed an upward trend, but compared with those of the study 1 group and study 2 group, the TCM symptom scores of the control group increased significantly and the difference between the three groups was statistically significant (P < 0.05). The effective rates of the control group, study group 1, and study group 2 were 42.11%, 82.05%, and 62.86%, respectively; compared with that of the control group, the treatment efficiency of study groups 1 and 2 was higher and the difference between the three groups was statistically significant (P < 0.05). Conclusion: Tropisetron hydrochloride is an effective treatment for patients with lung cancer before chemotherapy, which can effectively improve the side effects of nausea and vomiting caused by chemotherapy, reduce the white blood cell count, and improve the quality of life of patients; it plays an important role in the improvement of prognosis.

The effects and the mechanisms of naringenin from Artemisia ordosica Krasch on allergic rhinitis based on mast cell degranulation model and network pharmacology.

OBJECTIVES: The ethyl acetate extraction of Artemisia ordosica Krasch (AOK) root showed anti-allergic rhinitis (AR) effect, while the active compounds and pharmacological targets were unknown. METHODS: The P815 degranulation was established by cell counting kit 8 assay, ß-hexosaminidase releasing assay and toluidine blue staining. The flavonoids were screened in vitro. Then toluidine blue staining and ELISA were carried out to investigate the anti-inflammatory effects of the active compound. Network pharmacology was implemented to explain the mechanisms of the active compound. iGEMDOCK was used to investigate the binding between active compound and hub targets. KEY FINDINGS: C48/80 was the optimum reagent in triggering P815 degranulation. Naringenin could significantly decrease P815 degranulation. Meanwhile, naringenin could remarkably increase the IL-4 and decrease the tumour necrosis factor-α. The effect of naringenin on AR was achieved by regulating multiple targets (e.g. AKT1, MAPK3, VEGFA) and pathways (e.g. pathways in cancer, VEGF signalling pathway). Nine hub proteins were obtained by topological analysis. Multiple hydrogen bonds and van der Waals forces were formed between the naringenin and the residues of hub proteins. CONCLUSIONS: Naringenin might be one of the effective ingredients of AOK against AR. And its effects could achieve through regulating multiple targets and pathways.

TCQA, A Natural Caffeoylquinic Acid Derivative Attenuates H2O2-Induced Neuronal Apoptosis by Suppressing Phosphorylation of MAPKs Signaling Pathway.

1,3,5-Tri-O-caffeoyl quinic acid is a caffeoylquinic acid derivative isolated from the roots of Arctium lappa L. Our previous studies have revealed that the ethyl acetate extract of the roots of A. lappa L. and the caffeoylquinic acids contained in it possess antioxidant properties, especially 1,3,5-tri-O-caffeoyl quinic acid. The present study aimed to investigate the protective effects of 1,3,5-tri-O-caffeoyl quinic acid against hydrogen peroxide-induced oxidative stress and explore the underlying mechanism. We found that 1,3,5-tri-O-caffeoyl quinic acid prevented the decline of cell viability and excessive release of lactate dehydrogenase induced by hydrogen peroxide. In addition, Hoechst 33 342 staining and Annexin V-PI double staining showed that 1,3,5-tri-O-caffeoyl quinic acid inhibited hydrogen peroxide-induced neuronal cell apoptosis. 1,3,5-Tri-O-caffeoyl quinic acid reduced the excessive production of intracellular reactive oxygen species, decreased the malondialdehyde content, and improved the activity of superoxide dismutase. Furthermore, 1,3,5-tri-O-caffeoyl quinic acid restored the loss of mitochondrial membrane potential in SH-SY5Y cells induced by hydrogen peroxide. 1,3,5-Tri-O-caffeoyl quinic acid downregulated the overexpression of proapoptotic proteins, including Bax, cytochrome c, cleaved caspase-9, and cleaved caspase-3 as well as promoted the expression of the antiapoptotic protein Bcl-2. Moreover, the phosphorylation of mitogen-activated protein kinases induced by hydrogen peroxide was inhibited by 1,3,5-tri-O-caffeoyl quinic acid. Pretreatment with 1,3,5-tri-O-caffeoyl quinic acid also promoted the activation of phosphorylated Akt. Taken together, these findings suggest that 1,3,5-tri-O-caffeoyl quinic acid exerts protective effects against hydrogen peroxide-induced neuronal apoptosis. In addition, inhibition of the mitogen-activated protein kinase signaling pathway and the activation of Akt are implicated in the antioxidant activity of 1,3,5-tri-O-caffeoyl quinic acid, giving new insight in searching for a compound with antioxidant activity for the treatment of oxidative stress-associated neurological diseases.

Does coffee, tea and caffeine consumption reduce the risk of incident breast cancer? A systematic review and network meta-analysis.

OBJECTIVE: We aimed to evaluate the association between coffee and/or tea consumption and breast cancer (BC) risk among premenopausal and postmenopausal women and to conduct a network meta-analysis. DESIGN: Systematic review and network meta-analysis. SETTING: We conducted a systematic review of electronic publications in the last 30 years to identify case-control studies or prospective cohort studies that evaluated the effects of coffee and tea intake. RESULTS: Forty-five studies that included more than 3 323 288 participants were eligible for analysis. Network meta-analysis was performed to determine the effects of coffee and/or tea consumption on reducing BC risk in a dose-dependent manner and differences in coffee/tea type, menopause status, hormone receptor and the BMI in subgroup and meta-regression analyses. According to the first pairwise meta-analysis, low-dose coffee intake and high-dose tea intake may exhibit efficacy in preventing ER(estrogen receptor)- BC, particularly in postmenopausal women. Then, we performed another pairwise and network meta-analysis and determined that the recommended daily doses were 2-3 cups/d of coffee or ≥5 cups/d of tea, which contained a high concentration of caffeine, particularly in postmenopausal women. CONCLUSIONS: Coffee and tea consumption is not associated with a reduction in the overall BC risk in postmenopausal women and is associated with a potentially lower risk of ER- BC. And the highest recommended dose is 2-3 cups of coffee/d or ≥5 cups of tea/d. They are potentially useful dietary protectants for preventing BC.

Study on the experimental verification and regulatory mechanism of Rougui-Ganjiang herb-pair for the actions of thermogenesis in brown adipose tissue based on network pharmacology.

ETHNOPHARMACOLOGICAL RELEVANCE: Cinnamomum cassia Presl (Rougui) has character of xin、gan、wen, belongs to Jing of heart、lung、bladder, and has the effect of dispersing cold and relieving pain. It is widely used to resolve the exterior and dissipate cold in Treatise on Febrile Diseases (Shang Han Lun), such as Chaihu Guizhi Ganjiang Tang and Guizhi Renshen Tang. Both these two prescriptions contain Cinnamomum cassia Presl and Zingiber officinale Rosc (Ganjiang). Rougui-Ganjiang herb-pair (RGHP) can warm viscera and remove cold, which is widely used in Shang Han Lun. And in modern times, recent studies have showed that cinnamon and ginger also have the effect of thermogenesis and regulating the body temperature, respectively. AIM OF THE STUDY: To maintain the body thermal homeostasis and prevent cold invasion of main organs, in this study, we assessed the underlying physiological changes induced by RGHP in mice exposed to -20 °C and explored the mechanisms for the thermogenic actions of RGHP in brown adipose tissue (BAT) by network pharmacology and molecular docking. MATERIALS AND METHODS: Male Kunming (KM) mice were fed normal diet with orally administration of distilled water or ethanol RGHP extract (three doses: 375,750 and 1500 mg/kg) for 21 days, once per day and then exposed to -20 °C for 2 h. The core temperature, activity ability and the degree of frostbite in mice, morphological and ATP content of adipocytes were measured. In addition, the network pharmacology was employed to predict the targets of RGHP' s thermogenesis effect on BAT. Pathway analysis and biological process with key genes was carried out through KEGG and GO analysis, respectively. Furthermore, the core ingredients and targets obtained by network pharmacology were verified by molecular docking and Western blot assays. RESULTS: RGHP can significantly increase the core body temperature, reduce the degree of frostbite and enhance the activity ability of mice after cold exposure. Meanwhile, it can also improve the lipid morphology and decrease ATP production in BAT. A network pharmacology-based analysis identified 246 ingredients from RGHP (two herbs), which related to 222 target genes. There were 8 common genes between 222 compounds target genes and 62 thermogenesis associated target genes, which linked to 49 potential compounds. There are 24 ingredients which degree are greater than the average. Among them, we found that oleic acid, EIC, 6-gingerol, eugenol, isohomogenol and sitogluside could be detected in mice plasma. The cAMP-PPAR signaling pathway was enriched for thermogenesis after KEGG analysis with 8 genes. Molecular docking analysis and Western blot assay further confirmed that oleic acid, 6-gingerol, eugenol and isohomogenol were potential active ingredients for RGHP's heat production effect. And UCP1, PGC-1α, PPARα and PPARγ are key thermogenesis proteins. CONCLUSIONS: RGHP treatment can significantly maintain the rectal temperature of mice by enhancing the BAT heat production. RGHP exhibited the heat production effect, which might be mainly attributed to increasing thermogenesis through the cAMP-PPAR signaling pathway in cold exposure mice. Oleic acid, 6-gingerol, eugenol and isohomogenol might be considered the potential therapeutic ingredients which affect the key targets of thermogenesis effect.

Arctium lappa L. roots ameliorates cerebral ischemia through inhibiting neuronal apoptosis and suppressing AMPK/mTOR-mediated autophagy.

BACKGROUND: Arctium lappa L. roots are very popular cultivated vegetables, which possesses various pharmacological activities. Our previous studies have demonstrated that Arctium lappa L. roots exerted protective effects against H2O2, glutamate and N-methyl-D-aspartic acid (NMDA)-induced neuronal injury in vitro. However, whether Arctium lappa L. roots could prevent against cerebral ischemia and the underlying mechanism remain unclear. PURPOSE: The objective of the present study was to investigate the neuroprotective effects of ethyl acetate extract of Arctium lappa L. roots (EAL) and the active ingredient 4,5-O-dicaffeoyl-1-O-[4-malic acid methyl ester]-quinic acid (DCMQA) in EAL against cerebral ischemia and explore the underlying mechanism. STUDY DESIGN: The neuroprotective effects of EAL and DCMQA were investigated in rats with permanent middle cerebral artery occlusion (MCAO) and in oxygen glucose deprivation/reoxygenation (OGD/R)-stimulated SH-SY5Y cells, respectively. METHODS: The infarct volume, brain edema and neurological deficits were measured following MCAO. TUNEL and Nissl staining were performed to detect neuronal loss and apoptosis of neurons in rat brains. Cell survival was measured by MTT and LDH assay. In addition, reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) levels were determined by DCFH-DA and JC-1 fluorescent probe, respectively. Hoechst 33342 staining and Annexin V-FITC/PI double staining were performed to evaluate neuronal apoptosis. The expression levels of proteins were evaluated by western blot. RESULTS: EAL reduced brain infarct volume, ameliorated brain edema and improved neurological deficits in MCAO rats. In addition, EAL inhibited oxidative stress and inflammatory responses following MCAO. Besides, active compound DCMQA alleviated cytotoxicity as well as inhibited over-production of intracellular ROS and loss of MMP induced by OGD/R in SH-SY5Y cells. Moreover, EAL and DCMQA inhibited apoptosis by decreasing the expressions of pro-apoptotic proteins including bax, cytochrome c and cleaved caspase-3 while promoting the bcl-2 expression in MCAO rats and OGD/R-stimulated neurons, respectively. In addition, DCMQA suppressed the production of autophagosomes and down-regulated expression of Beclin 1 and LC3. Furthermore, inhibiting AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) signaling pathway contributed to DCMQA-mediated suppression of autophagy induced by OGD/R. CONCLUSION: Our findings demonstrate that Arctium lappa L. roots protect against cerebral ischemia through inhibiting apoptosis and AMPK/mTOR-mediated autophagy in vitro and in vivo, providing a theoretical basis for the development of CQAs in Arctium lappa L. roots as neuroprotective drugs for the prevention and treatment of ischemic stroke.

Network pharmacology-based research uncovers cold resistance and thermogenesis mechanism of Cinnamomum cassia.

BACKGROUND: Cinnamomum cassia (L.) J.Presl (Cinnamon) was known as a kind of hot herb, improved circulation and warmed the body. However, the active components and mechanisms of dispelling cold remain unknown. METHODS: The effects of several Chinses herbs on thermogenesis were evaluated on body temperature and activation of brown adipose tissue. After confirming the effect, the components of cinnamon were identified using HPLC-Q-TOF/MS and screened with databases. The targets of components were obtained with TCMSP, SymMap, Swiss and STITCH databases. Thermogenesis genes were predicted with DisGeNET and GeneCards databases. The protein-protein interaction network was constructed with Cytoscape 3.7.1 software. GO enrichment analysis was accomplished with STRING databases. KEGG pathway analysis was established with Omicshare tools. The top 20 targets for four compounds were obtained according to the number of edges of PPI network. In addition, the network results were verified with experimental research for the effects of extracts and major compounds. RESULTS: Cinnamon extract significantly upregulated the body temperature during cold exposure.121 components were identified in HPLC-Q-TOF/MS. Among them, 60 compounds were included in the databases. 116 targets were obtained for the compounds, and 41 genes were related to thermogenesis. The network results revealed that 27 active ingredients and 39 target genes. Through the KEGG analysis, the top 3 pathways were PPAR signaling pathway, AMPK signaling pathway, thermogenesis pathway. The thermogenic protein PPARγ, UCP1 and PGC1-α was included in the critical targets of four major compounds. The three major compounds increased the lipid consumption and activated the brown adipocyte. They also upregulated the expression of UCP1, PGC1-α and pHSL, especially 2-methoxycinnamaldehyde was confirmed the effect for the first time. Furthermore, cinnamaldehyde and cinnamon extract activated the expression of TRPA1 on DRG cells. CONCLUSION: The mechanisms of cinnamon on cold resistance were investigated with network pharmacology and experiment validation. This work provided research direction to support the traditional applications of thermogenesis.

Cinnamomum cassia extract promotes thermogenesis during exposure to cold via activation of brown adipose tissue.

ETHNOPHARMACOLOGICAL RELEVANCE: Cinnamomum cassia (L.) J.Presl (Lauraceae), a widely used traditional Chinese medicine, is well known to exert hot property. It is recorded as dispelling cold drug in ancient Chinese monographs, such as Synopsis of golden chamber published in Han dynasty. According to Chinese Pharmacopoeia (2015), Cinnamomum cassia (L.) J.Presl (Cinnamon) has the functions of dispersing cold, relieving pain, warming meridians and promoting blood circulation. AIM OF THE STUDY: The aim of this study is to evaluate the effect of Cinnamon extract (CE) on cold endurance and the mechanism of thermogenesis activity. MATERIALS AND METHODS: The improving effect of hypothermia were evaluated with body temperature by infrared camera and multi-thermo thermometer. In vivo, the thermogenic effect was observed with energy metabolism and substrate utilization. The activation of brown adipose tissue (BAT) was evaluated with the histomorphology and expression of thermogenic protein. In vitro, the uncoupling effect on mitochondrial was evaluated with Seahorse and fluorescent staining. The mechanism of thermogenesis was explored in brown adipocyte. RESULTS: The body temperature and energy expenditure were significantly increased by CE administration in cold environment. In morphology, lipid droplets were reduced and the number of mitochondrial was increased. CE significantly increased the non-shivering thermogenesis via upregulating the expression of thermogenic protein. In vitro, the uncoupling effect was obviously along with the decreased mitochondrial membrane potential and ATP production. It was confirmed that the thermogenesis effect was induced via lipolysis and energy metabolism. In addition, CE also alleviated myocardium injury in the morphology in cold environment. Moreover, the major constituent was identified as (1) coumarin, (2) cinnamic acid, (3) cinnamaldehyde and (4) 2-methoxy cinnamaldehyde. CONCLUSIONS: The mechanism of improving cold tolerance was related to lipolysis and activation of BAT. Meanwhile, we provided a kind of potential prevention methods for cold injury.

Kukoamine A Protects against NMDA-Induced Neurotoxicity Accompanied with Down-Regulation of GluN2B-Containing NMDA Receptors and Phosphorylation of PI3K/Akt/GSK-3ß Signaling Pathway in Cultured Primary Cortical Neurons.

Kukoamine (KuA) is a spermine alkaloid present in traditional Chinese medicine Cortex Lycii radices, which possesses various pharmacological properties. Our previous studies have demonstrated that KuA exerts neuroprotective effects against H2O2-induced oxidative stress, radiation-induced neuroinflammation, oxidative stress and neuronal apoptosis, as well as neurotoxin-induced Parkinson's disease through apoptosis inhibition and autophagy enhancement. The present study aimed to investigate the neuroprotective effects of KuA against NMDA-induced neuronal injury in cultured primary cortical neurons and explore the underlying mechanism. Incubation with 200 µM NMDA for 30 min induced excitotoxicity in primary cultured cortical neurons. The results demonstrated that pretreatment with KuA attenuated NMDA induced cell injury, LDH leakage and neuronal apoptosis. KuA also regulated apoptosis-related proteins. Thus, incubation with the alkaloid decreased the ratio of Bax/Bcl-2, and inhibited the release of cytochrome C, the expression of p53 and the cleavage of caspase-3. Moreover, KuA prevented the upregulation of GluN2B-containing NMDA receptors (NMDAR). Additionally, pretreatment with KuA reversed NMDA-induced dephosphorylation of Akt and GSK-3ß and the protective effect of KuA on NMDA-induced cytotoxicity was abolished by wortmannin, a PI3K inhibitor. Taken together, these results indicated that KuA exerted neuroprotective effects against NMDA-induced neurotoxicity in cultural primary cortical neurons and caused the down-regulation of GluN2B-containing NMDARs as well as the phosphorylation of proteins belonging to the PI3K/Akt/GSK-3ß signaling pathway.

Current Advances in Naturally Occurring Caffeoylquinic Acids: Structure, Bioactivity, and Synthesis.

Caffeoylquinic acids (CQAs) are a broad class of secondary metabolites that have been found in edible and medicinal plants from various families. It has been 100 years since the discovery of chlorogenic acid in 1920. In recent years, a number of naturally derived CQAs have been isolated and structurally elucidated. Accumulated evidence demonstrate that CQAs have a wide range of biological activities, such as antioxidation, antibacterial, antiparasitic, neuroprotective, anti-inflammatory, anticancer, antiviral, and antidiabetic effects. Up to date, some meaningful progresses on the biosynthesis and total synthesis of CQAs have also been made. Therefore, it is necessary to comprehensively summarize the structure, biological activity, biosynthesis, and chemical synthesis of CQAs. This review provides extensive coverage of naturally occurring CQAs discovered from 1990 until 2020. Modern isolation techniques, chemical data (including structure, biosynthesis, and total synthesis), and bioactivity are summarized. This would be helpful for further research of CQAs as potential pharmaceutical agents.

Notopterygium incisum extract (NRE) rescues cognitive deficits in APP/PS1 Alzhneimer's disease mice by attenuating amyloid-beta, tau, and neuroinflammation pathology.

ETHNOPHARMACOLOGICAL RELEVANCE: Alzheimer's disease (AD) is a frequently occurring disease of the elderly, and "deficiency" is the root of AD. Most famous experts of traditional Chinese medicine believe that the disease is based on deficiency, and the deficiency of kidney essence is the basis. Notopterygium incisum (Qiang huo) is beneficial to bladder, liver, and kidneys. It is used to treat liver and kidney deficiency, language difficulties, and mental coma. Qiang huo yu feng tang has been used to treat liver and kidney deficiency, unclear language and mental paralysis in many traditional Chinese medicine books and records. In modern times, it has been used to treat AD and exhibited favourable efficacy. AIM OF THE STUDY: This study attempts to investigate the effects of furocoumarins from Notopterygium incisum (NRE) on the Aß cascade, tau pathology and inflammatory pathology of AD. MATERIALS AND METHODS: In this study, we reported a detailed protocol for stabilizing HEK APPswe293T cells with lentivirus for the first time. This cell line can secrete high concentration of Aß. In addition, we treated N2a cells with AKT/PKC specific inhibitors (wortmannin/GF-109203X) and established a tau pathological cell model (AKT/PKC N2a) by activating GSK3ß and triggering hyperphosphorylation of tau. The Aß levels and the expression of phosphorylated tau were detected by ELISA and Western blot. The cognitive ability of NRE on APP/PS1 mice was detected using a Morris water maze (MWM) assay and Aß contents were also evaluated. RESULTS: In HEK APPswe293T cells, NRE (10, 20, 40 µg/mL) significantly inhibited the secretion and production of Aß in dose dependent manner. In addition, NRE also suppressed the expression of phosphorylated tau in wortmannin/GF-109203X treated N2a cells. Furthermore, NRE ameliorated the cognitive impairment of APP/PS1 mice, and the contents of Aß, IL-1ß and TNF-α were significantly depressed in hippocampus and cortex. CONCLUSION: In conclusion, our results demonstrated that NRE has a potential anti-AD effect via the inhibition of the Aß cascade, tau pathology and neuroinflammation in vitro and in vivo.

Jiang Gui Fang activated interscapular brown adipose tissue and induced epididymal white adipose tissue browning through the PPARγ/SIRT1-PGC1α pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Gui Zhi Tang, a well-known Chinese herbal formula recorded in the Eastern Han Dynasty, has been widely used to treat exogenous cold for thousands of years. Recent studies have shown that Gui Zhi Tang has the effect of regulating the body temperature. Because of its effect on heat production, protecting vital organs of the body and avoiding damage from the cold environment, Jiang Gui Fang (JG) was obtained from the Department of Traditional Chinese Medicine at the General Hospital of Northern Theatre Command where it has been used clinically for many years and has exhibited favourable efficacy. Based on research on Gui Zhi Tang, the principles of traditional Chinese medicine and survey of a large number of studies, this empirical formula was developed. The composition of JG included Dried ginger, Cassia twig, and Liquorice in Gui Zhi Tang, which play a major role in the treatment of exogenous cold, and combined these components with other Chinese medicines, such as Pueraria, Spatholobus, Acanthopanacis cortex, Evodiae fructus, and Codonopsis pilosula. AIM OF THE STUDY: To promote the core body temperature and prevent invasion of the major organs from the cold environment, we studied the effect of JG on the core body temperature of mice and then explored its regulation of interscapular brown adipose tissue (iBAT) and epididymal white adipose tissue (eWAT) and the possible mechanism. Finally, we determined the phytochemical composition of JG that plays a role in heat production. MATERIALS AND METHODS: In vivo study, we performed a 4-week treatment of JG in acute cold environment at -20 °C and chronic cold exposure at 4 °C. The core temperature, adipose tissue weight, serum parameters, and morphological observation of adipocytes, liver and kidney were measured. Then we investigated the expression levels of adipogenic factors, thermogenic factors and lipoprotein. In vitro, we determined the lipid droplet content, ATP content, and the maximum oxygen consumption of mitochondria. RESULTS: JG treatment promoted core temperature, inhibited eWAT weight, protected liver, and reduced glucose and lipids in Kunming (KM) mice. JG also increased the expression of BAT-associated thermogenic factors, including uncoupling protein 1 (UCP1) and peroxisome proliferator-activated receptor γ coactivator-1α (PGC1α). The levels of the lipogenic factor peroxisome proliferate-activator receptor gamma (PPARγ) and the lipolytic protein hormone-sensitive triglyceride lipase (HSL) in eWAT were elevated. The results of H&E and immunohistochemistry showed that JG significantly reduced the size of iBAT and eWAT and increased the content of UCP1. In vitro, JG reduced the content of lipid droplets and ATP in brown fat cells. The maximum oxygen consumption capacity of mitochondria and the expression levels of UCP1, PGC1α and silent mating type information regulation 2 homologue 1 (SIRT1) were enhanced after JG treatment. CONCLUSIONS: In vivo and in vitro studies, the results demonstrated that JG obviously increased the core temperature of mice by activating iBAT and inducing eWAT browning, which proved the mechanism is closely related to the PPARγ/SIRT1- PGC1α pathway. In this paper, we will provide a reference for further study of iBAT activation and eWAT browning.

Identification of New Potent Human Uncoupling Protein 1 (UCP1) Agonists Using Virtual Screening and in vitro Approaches.

Recent studies suggested that activation of Uncoupling Protein 1 (UCP1) has become an appealing therapeutic strategy against obesity and diabetes. In our research, the 3D structure of UCP1 was constructed through homology modelling, refined through molecular dynamics simulation, and evaluated by Ramachandran plot, the molecular docking of UCP1 activators brought about the proposal of an interaction mode inside the UCP1 active site. Remarkably, Reside Lys126 formed hydrogen bond; residues Pro121, Val125, Tyr146, Tyr149 and Arg150 formed hydrophobic interaction, which are key amino acids within UCP1 site. Then a pharmacophore model was generated consisting of three hydrophobic groups, a negative center and an additional hydrophobic group. Pharmacophore-based virutal screening of Specs database yield 5 hits. In vitro assay indicated ZINC 04660290 significantly increased the protein expression of UCP1 and decreased the fat droplet in a dose-dependent manner. Besides, pharmacokinetic properties were predicted for those five compounds through ADME/T prediction. All of these will guide us to design new UCP1 activators for the treatment of obesity and diabetes.

Alpinia oxyphylla oil induces apoptosis of hepatocellular carcinoma cells via PI3K/Akt pathway in vitro and in vivo.

BACKGROUND: The anti-tumor properties of Alpinia oxyphylla Miquel (A. oxyphylla) extracts and their petroleum ether (PE) fractions have long attracted scientific attention. These extracts' anti-tumor activity and mechanisms in vivo are still unclear. This study was designed to investigate the anti-tumor activity and the underlying mechanism of PE's effect on hepatocellular carcinoma (HCC) in vitro and in vivo. MATERIALS AND METHOD: The anti-tumor activity of PE was evaluated by MTT assay and xenograft study. Mechanistic studies of PE were analyzed by Hoechst 33342 staining, Annexin V-FITC/PI double-staining assay, immunohistochemical staining and western blot assay. The toxicity of the PE treatment was verified by the levels of liver and kidney function in nude mice and the H&E staining of their liver and kidney tissues. RESULT: PE significantly inhibited the growth of HepG2, BEL-7402, SMMC-7721 and Hep3B cells in a concentration- and time-dependent manner. Specifically, PE inhibited the growth of Hep3B cells by inducing apoptosis. PE treatment at the doses of 0.25, 0.5 and 1 g/kg for 21 days caused a respective 35.7 percent, 49.3 percent and 58.8 percent inhibition of the tumor volume, and a 14.8 percent, 40.2 percent and 55.6 percent decrease in the tumor weight, respectively, as compared with the vehicle group in tumor-loaded mice in vivo. PE promoted the release of cytochrome c from mitochondria to cytosol in a concentration-dependent manner. The expression levels of BAX (p < 0.01), cleaved caspase-9 (p < 0.01) and cleaved caspase-3 (p < 0.05) were increased significantly in the PE-treated group at the dose of 1 g/kg; the expression level of BAX (p < 0.05) was increased significantly in the PE-treated group at the dose of 0.5 g/kg, and the expression level of Bcl-2 (p < 0.01) was decreased significantly in the PE-treated group in a concentration-dependent manner. Apoptosis was induced by PE through up-regulating the expression of PTEN, down-regulating the expression of PI3K and inhibiting the phosphorylation of Akt. The liver and kidney function of the plasma and the morphology of the liver and kidney were normal in each group. CONCLUSION: These findings suggested that PE exhibited anti-cancer efficacy on Hep3B cell in vitro and in vivo. The induction of apoptosis might be one mechanism that underlies PE's ability to combat cancer by inhibiting the PI3K/Akt pathway. PE has no obvious toxicity in vivo when it exerts anti-tumor effects and has the potential to develop into an alternative anti-cancer drug for HCC treatment.

Novel ADAM-17 inhibitor ZLDI-8 enhances the in vitro and in vivo chemotherapeutic effects of Sorafenib on hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) is one of the greatest life threats for Chinese people, and the prognosis of this malignancy is poor due to the strong chemotherapy resistance in patients. Notch pathway components mediate cell survival and epithelial-mesenchymal transition (EMT), and also participate in the induction of multi-drug resistance (MDR). In the present study, we demonstrated the discovery of a novel inhibitor for Notch activating/cleaving enzyme ADAM-17, named ZLDI-8; it inhibited the cleavage of NOTCH protein, consequently decreased the expression of pro-survival/anti-apoptosis and EMT related proteins. ZLDI-8 treatment enhanced the susceptibility of HCC cells to a small molecular kinase inhibitor Sorafenib, and chemotherapy agents Etoposide and Paclitaxel. ZLDI-8 treatment enhanced the effect of Sorafenib on inhibiting tumor growth in nude HCC-bearing mice model. These results suggest that ZLDI-8 can be a promising therapeutic agent to enhance Sorafenib's anti-tumor effect and to overcome the MDR of HCC patients.

Can music improve sleep quality in adults with primary insomnia? A systematic review and network meta-analysis.

BACKGROUND: Primary insomnia is one of the most common issues for adults. However, whether to use music intervention as a non-pharmacological method of treatment, as well as which treatment should be preferred, is still a matter of controversy. Therefore, we aimed to compare and rank music interventions and no-music controls for primary insomnia patients. METHODS: A network meta-analysis was used to identify evidence from relevant clinical trials. We searched PubMed, Embase, the Cochrane Library, and the China National Knowledge Infrastructure Library for publications up to May 2017, pertaining to music intervention for primary insomnia patients. The prespecified primary outcome was sleep quality (scored by the PSQI and overall), and the secondary outcomes were sleep onset latency and sleep efficiency. We did pairwise meta-analyses using the random-effects model, later completing the random-effects network meta-analyses. The study was registered with PROSPERO, number CRD42017064750. RESULTS: We deemed 20 trials to be eligible, involving 1339 patients and 12 intervention arms. For PSQI scores, all intervention arms were statistically more effective than the usual care, with patients ranking listening to music as the best means of intervention (SMD: -0.61, 95%CrI: -1.01 to -0.20). For overall sleep quality, only music-associated relaxation was statistically more effective than the patients' usual care (-0.28, -0.48 to -0.08). In terms of sleep onset latency, music-associated relaxation and listening to music had significant advantages (-0.26, -0.64 to -0.09, and -0.28, -0.53 to -0.02); listening to music and music with exercise displayed a tendency to improve sleep efficiency. CONCLUSIONS: When considering the efficacy, music intervention seemed to offer clear advantages for adults with primary insomnia. Listening to music and music-associated relaxation are probably the best options to consider in the application of music intervention.