[Toxicity mechanism of Rhododendri Mollis Flos: based on serum metabolomics and network toxicology].

This study aims to explore the toxicity mechanism of Rhododendri Mollis Flos(RMF) based on serum metabolomics and network toxicology. The toxic effect of RMF on normal rats was evaluated according to the symptoms, serum biochemical indexes, and histopathology. Serum metabolomics was combined with multivariate statistical analysis to search endogenous differential metabolites and related metabolic pathways. The toxic components, targets, and signaling pathways of RMF were screened by network toxicology technique, and the component-target-metabolite-metabolic pathway network was established with the help of serum metabolomics. The result suggested the neurotoxicity, hepatotoxicity, and cardiotoxicity of RMF. A total of 31 differential metabolites and 10 main metabolic pathways were identified by serum metabolomics, and 11 toxic components, 332 related target genes and 141 main signaling pathways were screened out by network toxicology. Further analysis yielded 7 key toxic components: grayanotoxin Ⅲ,grayanotoxinⅠ, rhodojaponin Ⅱ, rhodojaponin Ⅴ, rhodojaponin Ⅵ, rhodojaponin Ⅶ, and kalmanol, which acted on the following 12 key targets: androgen receptor(AR), albumin(ALB), estrogen receptor ß(ESR2), sex-hormone binding globulin(SHBG), type 11 hydroxysteroid(17-beta) dehydrogenase(HSD17 B11), estrogen receptor α(ESR1), retinoic X receptor-gamma(RXRG), lactate dehydrogenase type C(LDHC), Aldo-keto reductase(AKR) 1 C family member 3(AKR1 C3), ATP binding cassette subfamily B member 1(ABCB1), UDP-glucuronosyltransferase 2 B7(UGT2 B7), and glutamate-ammonia ligase(GLUL). These targets interfered with the metabolism of gamma-aminobutyric acid, estriol, testosterone, retinoic acid, 2-oxobutyric acid, and affected 4 key metabolic pathways of alanine, aspartate and glutamate metabolism, cysteine and methionine metabolism, steroid hormone biosynthesis, and retinol metabolism. RMF exerts toxic effect on multiple systems through multiple components, targets, and pathways. Through the analysis of key toxic components, target genes, metabolites, and metabolic pathways, this study unveiled the mechanism of potential neurotoxicity, cardiotoxicity, and hepatotoxicity of RMF, which is expected to provide a clue for the basic research on toxic Chinese medicinals.

High-Expression of Neuropilin 1 Correlates to Estrogen-Induced Epithelial-Mesenchymal Transition of Endometrial Cells in Adenomyosis.

Epithelial-mesenchymal transition (EMT) induced by estrogen contributes to the development of adenomyosis. However, the exact underlying mechanism remains mostly obscure. We hypothesized that a transmembrane glycoprotein neuropilin 1 (NRP1) was critical in the EMT induced by estrogen, accelerating the development of adenomyosis. We firstly investigated the expression pattern of NRP1 in endometrium samples from women with adenomyosis. We found that NRP1 expression was significantly increased in the endometrium of uterine adenomyosis, especially in the ectopic endometrium. To determine the role of NRP1 in the EMT in endometrial cells, we used an NRP1 overexpression retrovirus to up-regulate the NPR1 expression in human endometrial cells (HEC-1-A). Endometrial cells infected with NRP1 retroviruses showed a high expression of NRP1 and exerted a mesenchymal phenotype, characterized by down-regulation of E-cadherin and Occludin, up-regulation of α-SMA and N-cadherin, and enhanced migration. Then, we found that 17ß-estradiol (E2) up-regulated the expression of NRP1 in endometrial cells in a dose-dependent manner, which was eliminated by raloxifene, a selective estrogen receptor inhibitor. Importantly, NRP1 shRNA significantly suppressed the EMT induced by E2 in endometrial cells. And NRP1 shRNA significantly inhibited the phosphorylation of Smad3 and restored the expressions of Slug and Snail1 mRNA. Collectively, these data highlight the possible role of NRP1 in the EMT in the development of adenomyosis and provide a potential therapeutic target for adenomyosis patients.

Development of the National Early Warning Score-Calcium Model for Predicting Adverse Outcomes in Patients With Acute Pancreatitis.

INTRODUCTION: This study aimed to develop a new model on the basis of the National Early Warning Score to predict intensive care unit admission and the mortality of patients with acute pancreatitis. METHODS: Patients diagnosed with acute pancreatitis in the emergency department were enrolled. The values of the National Early Warning Score, Modified Early Warning Score, and Bedside Index of Severity in Acute Pancreatitis in predicting intensive care unit admission and mortality of patients with acute pancreatitis were evaluated. RESULTS: A total of 379 patients with acute pancreatitis were enrolled; 77 patients (20.3%) were admitted to the intensive care unit and 14 (3.7%) died. The National Early Warning Score and calcium level were identified as independent risk factors of intensive care unit admission. Serum calcium exhibited a moderate correlation with National Early Warning Score (r = -0.46; P < 0.001), Modified Early Warning Score (r = -0.37; P < 0.001), and Bedside Index of Severity in Acute Pancreatitis (r = -0.39; P < 0.001). A new model called National Early Warning Score-calcium was developed by combining National Early Warning Score and calcium blood test result, which had larger areas under the curve for predicting intensive care unit admission and mortality than the other 3 scoring systems. DISCUSSION: A new model developed by combining National Early Warning Score and calcium exhibited better value in predicting the prognosis of acute pancreatitis than the models involving National Early Warning Score, Modified Early Warning Score, and Bedside Index of Severity in Acute Pancreatitis alone.

Rapid screening of drug-protein binding using high-performance affinity chromatography with columns containing immobilized human serum albumin.

For drug candidates, a plasma protein binding (PPB) more than 90% is more meaningful and deserves further investigation in development. In the study, a high-performance liquid chromatography method employing column containing immobilized human serum albumin (HSA) to screen in vitro PPB of leading compounds was established and successfully applied to tested compounds. Good correlation (a coefficient correlation of 0.96) was attained between the reciprocal values (X) of experimentally obtained retention time of reference compounds eluted through HSA column and the reported PPB values (Y) with a correlation equation of Y = 92.03 - 97.01X. The method was successfully applied to six test compounds, and the result was confirmed by the conventional ultrafiltration technique, and both yielded equal results. However, due to the particular protein immobilized to column, the method cannot be applied for all compounds and should be exploited judiciously based on the value of the logarithmic measure of the acid dissociation constant (pKa) as per the requirement. If α1-acid glycoprotein and other plasma proteins could be immobilized like HSA with their actual ratio in plasma to column simultaneously, the result attained using immobilized column may be more accurate, and the method could be applied to more compounds without pKa limitation.

2-methoxyjuglone induces apoptosis in HepG2 human hepatocellular carcinoma cells and exhibits in vivo antitumor activity in a H22 mouse hepatocellular carcinoma model.

In order to discover anticancer agents from natural sources, an ethanol-soluble extract of the root bark of Juglans cathayensis was investigated and showed cytotoxic effects against various human cancer cell lines. A subsequent phytochemical study on the EtOAc-soluble fraction determined 2-methoxyjuglone (1) as one of the main active constituents. Compound 1 was shown to be cytotoxic against HepG2 cells. Morphological features of apoptosis were observed in 1-treated HepG2 cells, including cell shrinkage, membrane blebbing, nuclear condensation, and apoptotic body formation. Cell cycle analysis with propidium iodide staining showed that 1 induced cell cycle arrest at the S phase in HepG2 cells. Flow cytometric analysis with annexin V and propidium iodide staining demonstrated that 1 induced HepG2 cell apoptotic events in a dose-dependent manner (0-8 µg/mL). Western blot analysis of apoptosis-related proteins revealed that 1 induces HepG2 cell apoptosis through mitochondrial cytochrome c-dependent activation of the caspase-9 and caspase-3 cascade pathway (intrinsic pathway). An in vivo experiment using tumor-bearing mice showed that treatment with 1 at 0.5 and 1.0 mg/kg per day decreased the tumor mass by 56% and 67%, respectively.

[Antipyretic effect of moxibustion at different temperatures and its relationship with the activity of temperature sensitive neurons in thermotaxic center].

OBJECTIVE: To discover the central mechanisms of antipyretic effect of moxibustion and its relationship with the acupoint sensor so as to provide the scientific evidence for "the treatment of heat syndrome with moxibustion". METHODS: Eighteen New Zealand Rabbits were randomly assigned into three groups, named group A (modeling with intravenous injection of Endotoxin), group B (moxibustion at 40 degrees C after Endotoxin injection) and group C (moxibustion at 48 degrees C after Endotoxin injection), 6 rabbits in each one. The experiment was undergoing in the condition of muscular relaxation and artificial respiration for the animals. The spotlight moxibustion at constant temperature was applied to "Zhiyang" (GV 9). The discharge of heat sensitive neurons (HSNs) at the preoptic region and anterior hypothalamus (POAH) was taken as the index. The impacts of the treatment on HSNs were observed in each group. RESULTS AND CONCLUSION: Moxibustion had significant antagonism to the pyrogen on its inhibition to the activity of HSNs in the thermotaxic center. As a result, the antipyretic effect was obtained. It is concluded that the effective result of moxibustion is achieved by stimulating polymodal receptors of acupoints.

[Studies on the chemical constituents of Toricellia angulata var. intermedia].

OBJECTIVE: To study the chemical constituents of Toricellia angulata var. intermedia. METHODS: The constituents were isolated and purified by repeated column chromatography and their structures were elucidated by spectroscopic analysis. RESULTS: Twelve compounds including beta-sitoterol (1), 7-hydroxy-3-ethylphthalide (2), 3beta-methoxy-stigmast-7-ene (3), stigmast-5-ene (4), trans-p-methylcinnamaldehyde (5), stigmate-7-en-3beta-ol (6), o. p-dimethoxybenzoicacid (7), beta-daucosterol (8), ursolicacid (9), stearic acid (10), docosanoic acid (11), palmitic acid (12) were isolated and identified from this plant. CONCLUSION: All the compounds are isolated from the plant for the first time, compounds 3 -7, 10 -12 are isolated from this genus for the first time.

[Effects of strong and weak electroacupuncture on endotoxin-induced changes of electrical activities of heat-sensitive neurons in preoptic area and anterior hypothalamus in rabbits].

OBJECTIVE: To observe the effects of weak and strong electroacupuncture (EA) on endotoxin (ET) thermolysis-induced changes of discharges of neurons in the preoptic region and anterior hypothalamus (PO-AH) so as to explore its underlying mechanism in antipyretic and thermolytic actions and its relation to the receptive system of acupoints. METHODS: Eighteen New Zealand rabbits were randomly divided into ET group, ET + weak-EA group and ET + strong-EA group. Extracellular discharges of the PO-AH neurons were recorded by using tungsten microelectrodes. A "U"-shape stainless steel tube was implanted in the region (P0.4-A4.4, L0.5-1.7) crossing the hypothalamus for changing local temperature by perfusion of cool (25 degrees C) or warm (41 degrees C) solution in order to distinguish the heat sensitive neurons (HSN), cold sensitive neurons (CSN) and insensitve neurons to temperature changes. Intravenous injection of endotoxin (25 EU/rabbit) was given to rabbits to induce increase of tempe rature. EA (8 Hz, wave width 0.1 ms, weak stimulation: 4.5 V, strong stimulation: 25 V) was applied to bilateral "Yongquan" (KI 1) for observing changes of firing rates of HSN in PO-AH. RESULTS: Compared with the basal values of firing rates of PO-AH neurons in each group, the average changing ratios of both ET and ET + weak-EA groups decreased significantly from 55-60 min on in ET group and from 40-45 min on in ET + weak-EA group after intravenous injection of ET (P<0.05), suggesting no marked effect of weak EA for preventing discharges of PO-AH neurons from decrease. While in ET + strong-EA group, the firing rates of HSN of PO-AH kept stable after injection of ET during EA and after cease of EA (P>0.05 vs basal value), suggesting that strong EA could antagonize ET thermolysis-induced decrease of firing rates of PO-AH neurons. CONCLUSION: Stronger EA stimulation of KI1 can antagonize ET thermolysis-induced effect on electrical activities of PO-AH HSN, which may be initiated by the activation of the high-threshold thin nerve fibers in the acupoint region.

[The role of p38 MAPK pathway in ischemia-reperfusion injury of isolated liver].

OBJECTIVE: To study the effect of p38 MAPK activity on isolated rabbit liver during the period of cold preservation and reperfusion. METHODS: Based on the cold preservation-reperfusion model of isolated rabbit livers, according to the concentration of SB202190 in the preservation solution which was a specific p38 MAPK inhibitor, the isolated livers were divided into 4 groups, six in each A, B, C and D. Liver tissue samples and blood samples were harvested at different time points: before and end of cold preservation, reperfusion for 5, 10, 15, 30, 60, and 120 minutes. The activity levels of p38 MAPK were detected by both western blot and immunoprecipitation. The function markers of isolated livers were detected with automatic biochemistry analyzer, and the levels of total bile after reperfusion were measured. RESULTS: In normal rabbit liver tissues, p38 MAPK had low activity. During the cold preservation period, the activity of p38 MAPK elevated slightly. But during the reperfusion period, the activity of p38 MAPK changed markedly which elevated rapidly at the early stage and reached its peak value at 10 minutes, then decreased gradually to the normal level (7.6 +/-0.9) at 120 minutes. SB202190 could inhibit the activity in a dose-dependent manner. The peak values of p38 MAPK activity in group B,C and D were 42.5 +/-2.4, 10.1+/-1.4, and 7.6 +/-0.6 respectively, while 78.6 +/-6.1 in group A. During the reperfusion period, the levels of serum ALT, AST and ALP were higher in group A than those in any other group, alike the p38 MAPK activity, especially at 15 and 30 minutes. On the other hand, the total bile secretion volume was (10.2 +/-2.9) ml/100 g, (13.9 +/-1.3) ml/100 g, (15.6 +/-1.2) ml/100 g, and (16.0 +/-1.3) ml/100 g in group A, B, C and D respectively. CONCLUSIONS: During the cold preservation- reperfusion period, p38 MAPK specific inhibitor SB202190 can lower the p38 MAPK activity, and ameliorate the isolated liver injury. Activation of p38 pathway is one of the important mechanisms to cause ischemia-reperfusion injury of isolated liver.