Elucidating the therapeutic mechanism of Hengqing II decoction in Alzheimer's disease using network pharmacology and molecular docking techniques.

PURPOSE: The aim of our research was to investigate the mechanism of the Hengqing II decoction in treating Alzheimer's disease (AD) through network pharmacology and experimental validation methods. METHODS: Firstly, the major chemical compounds of Hengqing II decoction were characterized by ultra-high-performance liquid chromatography-high resolution mass spectrometry (UHPLC-Q-TOF-MS/MS), and the gene sets related to AD treatment by Hengqing II decoction were collected through the database of PubChem, Swiss TargetPrediction, and DisGeNET. Secondly, a multi-level molecular network of "Traditional Chinese medicine (TCM)-compound-target-disease" was constructed and visualized using the STRING platform and Cytoscape 3.9.1 software, and the enrichment analysis based on the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway databases was performed to predict the potential active compounds and targets of Hengqing II decoction for treating AD. Finally, molecular docking simulation was applied to investigate the binding interactions between potential active compounds and key targets, and the western blotting technique was employed to examine the expression levels of AKT1, TNF-α, and NOS2 proteins affected by active compounds. RESULTS: Totally 120 compounds in Hengqing II decoction were characterized by UHPLC-Q-TOF-MS/MS. Network pharmacology results showed that potential active compounds in Hengqing II decoction in treating AD included catalpol, gastrodin, and rehmannioside D, etc., and the main target proteins were TNF-α, NOS2, and AKT1. Further functional enrichment analysis revealed that Hengqing II decoction mainly exerted its therapeutic effects on AD by regulating lipid and atherosclerosis signaling pathways, AD signaling pathways, AKT1 signaling pathways, and PTGS2 signaling pathways. CONCLUSION: Hengqing II decoction exerted therapeutic effects on AD through multi-component, multi-target, and multi-pathway regulation, and its action mechanisms were related to oxidative stress, neuroinflammation, autophagy, and other pathways. Our research laid the data foundation for further exploration of action mechanism and clarification of clinical positioning and provided new ideas and clues in TCM formula research.

Study on network pharmacology of Ginkgo biloba extract against ischaemic stroke mechanism and establishment of UPLC-MS/MS methods for simultaneous determination of 19 main active components.

INTRODUCTION: Ginkgo biloba extract (GBE) is an effective substance from traditional Chinese medicine (TCM) G. biloba for treating ischaemic stroke (IS). However, its active ingredients and mechanism of action remain unclear. OBJECTIVES: This study aimed to reveal the potential active component group and possible anti-IS mechanism of GBE. MATERIALS AND METHODS: The network pharmacology method was used to reveal the possible anti-IS mechanism of these active ingredients in GBE. An ultra-high-performance liquid chromatography triple quadrupole electrospray tandem mass spectrometry (UPLC-MS/MS) method was established for the simultaneous detection of the active ingredients of GBE. RESULTS: The active components of GBE anti-IS were screened by literature integration. Network pharmacology results showed that the anti-IS effect of GBE is achieved through key active components such as protocatechuic acid, bilobalide, ginkgolide A, and so on. Gene Ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that the possible anti-IS mechanism of GBE is regulating the PI3K-Akt signalling pathway and other signal pathways closely related to inflammatory response and apoptosis regulation combined with AKT1, MAPK, TNF, ALB, CASP3, and other protein targets. Nineteen main constituents in seven batches of GBE were successfully analysed using the established UPLC-MS/MS method, and the results showed that the content of protocatechuic acid, gallic acid, ginkgolide A, and so forth was relatively high, which was consistent with network pharmacology results, indicating that these ingredients may be the key active anti-IS ingredients of GBE. CONCLUSION: This study revealed the key active components and the anti-IS mechanism of GBE. It also provided a simple and sensitive method for the quality control of related preparations.

Investigations of the gingerol oil colon targeting pellets for the treatment of ulcerative colitis.

The clinical treatment of ulcerative colitis (UC) faces great challenges due to lifetime medication. In this study, Gingerol oil was extracted and purified by the process easily scale-up and cost effective, with productivity 2.72 ± 0.38% (w/w, versus crude drugs). The quality control of gingerol oil was fully established by HPLC fingerprint with 4 common peaks identified as 6-gingerol, 8-gingerol, 6-shogaol and 10-gingerol. The similarities of 6 batches of gingerol oil are within 0.931-0.999. The protective effects of gingerol oil are equivalent to or even stronger than that of 6-gingerol on inflammation and oxidative stress of HT-29 cells induced by lipopolysaccharide and H2O2, as well as on UC in mice caused by dextran sulfate sodium salt (DSS). Our research conclusions coincide well with the holistic view of Traditional Chinese Medicine and network pharmacology. The absorption kinetics of gingerol oil were conducted using the in situ intestinal perfusion in rats and comparable absorption were achieved in the jejunum, ileum and colon segments within 2 h. Thus, gingerol oil colon targeting pellets were prepared by extrusion-spherization technique. The cumulative dissolution behaviors and mechanisms were observed and analyzed by fitting to dissolution model. Our studies provided reliable theoretical and experimental support for the gingerol oil as reliable therapeutic choice of UC.

Porcine cardiac blood - Salvia miltiorrhiza root alleviates cerebral ischemia reperfusion injury by inhibiting oxidative stress induced apoptosis through PI3K/AKT/Bcl-2/Bax signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Salvia miltiorrhiza Bge. mixed with porcine cardiac blood (PCB-DS) is mainly employed for the treatment of brain ischemia-induced mental disturbances, palpitations and phlegm confusion based on the traditional principle of Menghe medical sect. PCB is the guide to DS and enhances the effect of DS. However, the potential mechanism of PCB-DS preventing cerebral ischemia/reperfusion injury (CIRI) from the perspective of oxidative stress induced cell apoptosis remains unknown. AIM OF THE STUDY: To investigate the pharmacological activity and molecular mechanism of PCB-DS against CIRI. MATERIALS AND METHODS: DS samples processed with different methods were prepared and UPLC-Q-TOF-MS/MS was employed for qualitative analysis of the respective processing product. The middle cerebral artery occlusion reperfusion model was then established to investigate the pharmacological activities of PCB-DS. Pathological changes in the rat brain were observed by triphenyl tetrazolium chloride (TTC), hematoxylin-eosin, and TUNEL staining. The levels of IL-6, IL-1ß, and TNF-α were detected by ELISA to evaluate the inflammatory damage. Metabolomics of cerebrospinal fluid was further used to explore the potential mechanism of PCB-DS in preventing CIRI. Based on this, the levels of oxidative stress-related lactate dehydrogenase (LDH), reactive oxygen species (ROS), malondialdehyde (MDA), and superoxide dismutase (SOD) were determined. The protein levels of PI3K, AKT, Bcl-2, Bax, cleaved-caspase-3, and cleaved-caspase-9 proteins of the cerebral infarct zone were finally measured by western blotting. RESULTS: Forty-seven components were identified in four processing products. Compared to DS, the content of total aqueous components in PCB-DS was significantly increased including salvianolic acid B isomer, salvianolic acid D, salvianolic acid F, and salvianolic acid H/I/J. Among the DS, DS processed with wine, DS processed with pig blood, and DS processed with porcine cardiac blood, PCB-DS best alleviated the CIRI through the neurological score, brain infarct volume, brain histopathology and the levels of inflammatory factors in the brain. Twenty-five significant metabolites in the cerebrospinal fluid were screened out between the sham and I/R groups. They were mainly involved in the beta-alanine metabolism, histidine metabolism, and lysine degradation, which indicated that PCB-DS may inhibit oxidative stress-induced apoptosis to achieve treating ischemic stroke. The results of biomedical examination showed that PCB-DS could alleviate oxidative damage, significantly downregulate the expression of Bax, cleaved caspase-3 and cleaved caspase-9, and upregulate the expression of p-PI3K, p-AKT, and Bcl-2. CONCLUSION: In summary, this study demonstrated that PCB-DS alleviated CIRI and the molecular mechanism may be related to inhibiting the oxidative stress induced apoptosis through PI3K/AKT/Bcl-2/Bax signaling pathway.

Influence of Management of Intensive Weight, Blood Pressure, and Lipids on Disease Severity in Patients with Carotid Atherosclerosis.

Context: Cardiovascular diseases (CVDs caused by atherosclerosis, such as coronary heart disease and stroke, have become major causes of death and disability worldwide. Atherosclerosis is the primary pathological factor causing CVDs. Managing weight, blood pressure, and lipids is one of the tenets of chronic-disease management, including atherosclerosis. Objective: The study intended to investigate the effects of managing weight, blood pressure, and lipids on disease severity in patients with carotid atherosclerosis. Design: The research team designed a randomized, controlled trial. Setting: The study took place in the pediatric department at the First Hospital of Hebei Medical University in Shijiazhuang, Hebei Province, China. Participants: Participants were 380 patients with carotid atherosclerosis who entered the hospital between March 2018 and June 2020. Intervention: Participants were randomly assigned, using the random-number-table method, to an intervention or a control group, with 190 participants in each group. Both groups received anti-atherosclerotic treatments, and the intervention group also took part in a program for combined management of weight, blood pressure, and blood lipids. Outcome Measures: All measurements occurred at baseline and postintervention. Using a questionnaire, the study measured the changes in the two groups related to alcohol consumption, smoking, high-fat diet, high-salt diet, and lack of exercise. A physical examination provided participants' weights, blood pressures, and lipid levels, and the Self-Care Ability Assessment Scale (ESCA) provided the changes in their self-management ability. A carotid-artery examination measured parameters related to carotid atherosclerosis, including intima-media thickness (IMT), Crouse scores, plaque-class scores, and plaque-grade scores. Results: At baseline, no statistically significant differences existed between the groups. Postintervention, the intervention group had significantly greater decreases than the control group for alcohol consumption, smoking, high-fat diet, high-salt diet, lack of exercise, weight, blood pressure, lipid levels, intima-media thickness (IMT) scores, Crouse scores, and plaque-grade scores. Postintervention, the intervention group had significantly greater increases than the control group for self-responsibility, health knowledge, self-concept, and self-care-skills scores. Conclusions: A program for management of body weight, blood pressure, and blood lipids can effectively control the severity of carotid atherosclerosis, can prevent the disease's progression, and can be promoted as a clinical application.

Factors Underlying the Prevalence of Pythium Infection of Corn Seeds Following Seed Treatment Application of Tebuconazole.

Microbial communities are essential for soil health, but fungicide application may have significant effects on their structure. It is difficult to predict whether nontarget pathogens of applied fungicides in the soil will cause crop damage. Tebuconazole is a triazole fungicide that can be used as a seed treatment and, thereby, introduced to the soil. However, seed-applied tebuconazole has a potential risk of causing poor emergence of corn (Zea mays) seedlings. Using soil with a history of poor corn seedling emergence, we demonstrate through TA cloning and isolation that the poor emergence of corn seedlings from tebuconazole-coated corn seeds was primarily because of infection by surviving soil pathogens, specifically Pythium species that are not targeted by tebuconazole, rather than the phytotoxic effects of tebuconazole. Bioassay tests on tebuconazole-amended media showed that tebuconazole can suppress soil fungi while allowing Pythium to grow. Pythium species primarily contributing to the corn seed rot were more pathogenic at cooler temperatures. Furthermore, the nontarget biocontrol agent of Trichoderma spp. was strongly inhibited by tebuconazole. Taken together, the nontarget effects of tebuconazole are likely not significant under favorable plant growing conditions but are considerable because of low-temperature stress.

Radix Kansui Stir-Fried with Vinegar Reduces Radix Kansui-Related Hepatotoxicity in Mice via Mitochondrial Pathway.

OBJECTIVE: To investigate the mechanism of Radix Kansui (RK) stir-fried with vinegar (VRK) decreased hepatotoxicity in mice. METHODS: According to a random number table, 40 mice were randomly divided into negative control group (0.5% carboxymethylcellulose sodium, 20 mL/kg), positive control group (0.1% mixture of carbon tetrachloride in soybean oil, 20 mL/kg), RK group (the ethyl acetate extracts of RK, 250 g crude drug/kg) and VRK group (the ethyl acetate extracts of VRK, 250 g crude drug/kg) with 10 mice per group. All mice were administered orally by gavage daily for 7 continuous days. The morphology of liver tissues was examined to assess the liver injury by a transmission electron microscope. Hepatocyte apoptosis in vivo was determined by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nickend labeling (TUNEL) assay. Immunohistochemical technique was adopted to detect the expression of particular antiapoptotic and proapoptotic proteins in the mitochondrial pathways, including B-cell lymphoma (Bcl-2) and caspase-3, as well as the expression of inflammatory mediators, including nuclear factor kappa B (NF- κ B) and intercellular adhesion molecule-1 (ICAM-1). RESULTS: Liver injury and hepatocyte apoptosis were observed in RK mice, and the liver injury were significantly reduced in VRK-treated mice. In immunohistochemistry study, compared with the negative control group, RK inhibited dramatically the Bcl-2 protein expression and significantly increased the expression of caspase-3, NF- κ B and ICAM-1 (all P<0.01). Compared with the RK group, VRK group induced significant increase on Bcl-2 protein expression, and decreased the caspase-3, NF- κ B and ICAM-1 protein expression (P<0.05 or P<0.01). CONCLUSION: The mechanism of reduced hepatotoxicity of VRK may be associated with the reduced inflammation, regulation of antiapoptotic and proapoptotic mediators in the mitochondrial pathway.

Integrated Proteomics, Biological Functional Assessments, and Metabolomics Reveal Toosendanin-Induced Hepatic Energy Metabolic Disorders.

Toosendanin (TSN), a compound from Melia toosendan, exhibits severe hepatotoxicity, which restricts its clinical application. However, the mechanism is not clear. Our previous research found that covalent modification of TSN for proteins might be a possible reason using human liver microsomes, and the glycolytic enzymes, triosephosphate isomerase 1 (TPIS) and α-enolase (ENOA), were responsible for the hepatotoxicity. In this study, we tried to prove these findings in cell and animal models by integration of proteomics, metabolomics, and biological methods. Proteomics analysis in rats showed that TPIS and ENOA were covalently modified by TSN reactive metabolites. The biological functional assessments revealed that the modifications inhibited the activity of TPIS and induced the activity of ENOA, in vitro and in vivo, followed by an increase in the level of cellular methylglyoxal, advanced glycation end products, and reactive oxygen species/superoxide, and the induction of mitochondrial dysfunction, which further inhibited oxidative phosphorylation and stimulated glycolysis. Furthermore, metabolomics demonstrated the decrease in the level of metabolites in the tricarboxylic acid cycle, fatty acid ß-oxidation, and amino acid metabolism; i.e., TSN induced hepatocyte energy metabolism disorder. In conclusion, these data suggest novel mechanistic insights into TSN-induced liver injury on the upstream level and provide valuable proteins and energy metabolic targets for diagnosis and therapy in the clinic.

Potentially Cardiotoxic Diterpenoid Alkaloids from the Roots of Aconitum carmichaelii.

Aconitum carmichaelii is a traditional Chinese herbal medicine used for the treatment of pain and inflammation in the joints. However, the strong cardiotoxicity hinders its use. Although diester- and monoester-type diterpenoids, e.g., aconitine, mesaconitine, and hypacaonitine, are commonly considered as the toxic components, the toxicity of A. carmichaelii cannot be completely explained by the compounds reported. To investigate further the cardiotoxic compounds and their potential mechanism, the chemical constituents were first isolated by column chromatography and identified using mass spectrometry and NMR spectroscopy. Two new hetisine-type (1 and 2) and four new aconitine-type alkaloids (3-6) were assigned. The cardiac cytotoxicity assessed on H9c2 cells indicated that the new compound 4 as well as six known alkaloids (7 and 9-13) exhibited significant toxicities. A preliminary structure-toxicity relationship study suggested that substitution at C-8 and C-10 both have a significant influence on cardiotoxicity, and such toxicity decreased in the order OBz-8, OBu-8, and OMe-8. The presence of an OH-10 group abolished the toxicity. Finally, it was found that ion channel disorder and induction of mitochondrial-mediated cell apoptosis are the possible mechanisms of cardiotoxicity among the compounds studied.

[Historical evolution and modern research progress on characteristic processing of porcine cardiac blood processed Salvia miltiorrhiza in Menghe medical school].

The porcine cardiac blood processed Salvia miltiorrhiza (PCB Danshen) is the characteristic processing of Menghe medical school and has been inherited for hundreds of years, commonly used in the treatment of brain ischemia-induced agitation, palpitation and phlegm confusing heart. Ancient and modern physicians believe that porcine cardiac blood is a guiding for heart nourishing drugs, which could enhance the effects of Salvia miltiorrhiza by nourishing and soothing the nerves. However, the material basis and processing mechanism of PCB Danshen are still unclear. This paper investigated the historical evolution and modern research of PCB Danshen, including the clinical application, the intention of clinic processing, the processing technology and recent research of PCB Danshen. In addition, the major problems and significance in research and development of PCB Danshen were further thought and prospected, hoping to provide basic data for material basis and processing mechanism of PCB Danshen, and provide effective support for inheriting and carrying forward the characteristic processing technology of Menghe medical school.

The component formula of Salvia miltiorrhiza and Panax ginseng induces apoptosis and inhibits cell invasion and migration through targeting PTEN in lung cancer cells.

Lung cancer still remains the leading cause of cancer-related death worldwide. It is an urgent need for development of novel therapeutic agents to improve current treatment of this disease. Here we investigate whether the effective component formula of traditional Chinese Medicine could serve as new potential therapeutic drugs to treat lung cancer. We optimize the most effective component formula of Salvia miltiorrhiza and Panax Ginseng (FMG), which is composed of Salvianolic acid A, 20(S)-Ginsenoside and Ginseng polysaccharide. We discovered that FMG selectively inhibited lung cancer cell proliferation and induced apoptosis but had no any cytotoxic effects on normal lung epithelial BEAS-2B cells. Moreover, FMG inhibited lung cancer cell migration and invasion. Mechanistically, we found that FMG significantly promoted p-PTEN expression and subsequently inhibited PI3K/AKT signaling pathway. The phosphatase activity of PTEN protein was increased after FMG bound to PTEN protein, indicating that PTEN is one of the FMG targeted proteins. In addition, FMG regulated expression of some marker proteins relevant to cell apoptosis, migration and invasion. Collectively, these results provide mechanistic insight into the anti-NSCLC of FMG by enhancing the phosphatase activity of PTEN, and suggest that FMG could be as a potential option for lung cancer treatment.

An Ingenol Derived from Euphorbia kansui Induces Hepatocyte Cytotoxicity by Triggering G0/G1 Cell Cycle Arrest and Regulating the Mitochondrial Apoptosis Pathway in Vitro.

Natural product lingenol, a purified diterpenoid compound derived from the root of Euphorbia kansui, exerts serious hepatotoxicity; however, the molecular mechanisms remain to be defined. In the present study, cell counting Kit-8 (CCK-8), inverted phase contrast microscope and flow cytometry were used to demonstrate that lingenol significantly inhibited L-O2 cells proliferation, and induced cell cycle arrest and apoptosis. Moreover, the results investigated that lingenol markedly disrupted mitochondrial functions by high content screening (HCS). In addition, the up-regulation of cytochrome c, AIF and Apaf-1 and activation of caspases were found in L-O2 cells detected by Western blotting and ELISA assay, which was required for lingenol activation of cytochrome c-mediated caspase cascades and AIF-mediated DNA damage. Mechanistic investigations revealed that lingenol significantly down-regulated the Bcl-2/Bax ratio and enhanced the reactive oxygen species (ROS) in L-O2 cells. These data collectively indicated that lingenol modulation of ROS and Bcl-2/Bax ratio led to cell cycle arrest and mitochondrial-mediated apoptosis in L-O2 cells in vitro. All of these results will be helpful to reveal the hepatotoxicity mechanism of Euphorbia kansui and to effectively guide safer and better clinical application of this herb.

Antihepatocellular Carcinoma Potential of Tetramethylpyrazine Induces Cell Cycle Modulation and Mitochondrial-Dependent Apoptosis: Regulation of p53 Signaling Pathway in HepG2 Cells In Vitro.

Tetramethylpyrazine (TMP) was originally isolated from a traditional Chinese herbal medicine, Ligusticum chuanxiong In the present study, TMP exhibits potent antitumor activities in vitro. However, the molecular mechanisms remain to be defined. Hence, this study aims to investigate the antiproliferative and apoptotic effects of TMP on HepG2 and elucidate the underlying mechanisms. Analyses using Cell Counting Kit-8 and real-time cell analyzer indicated that TMP significantly inhibited HepG2 cell proliferation. We also observed that TMP induced cell cycle arrest at the G0/G1 checkpoint and apoptosis, using flow cytometry and high-content screening. Furthermore, our results predicted that TMP could directly decrease mitochondrial membrane potential (Δψm), increase the release of cytochrome c, and increase caspase activation, indicating that mitochondrial pathway apoptosis could be the mechanism for TMP within HepG2 cells. Moreover, TMP altered expression of p53 and the Bcl-2/Bax protein ratio, which revealed that TMP induced cell cycle arrest and caspase-dependent mitochondrial apoptosis in HepG2 cells in vitro. These studies provided mechanistic insights into the antitumor properties of TMP, which may be explored as a potential option for treatment of hepatocellular carcinoma.

Combretastatin A-4 and Derivatives: Potential Fungicides Targeting Fungal Tubulin.

Combretastatin A-4, first isolated from the African willow tree Combretum caffrum, is a tubulin polymerization inhibitor in medicine. It was first postulated as a potential fungicide targeting fungal tubulin for plant disease control in this study. Combretastatin A-4 and its derivatives were synthesized and tested against Rhizoctonia solani and Pyricularia oryzae. Several compounds have EC50 values similar to or better than that of isoprothiolane, which is widely used for rice disease control. Structure-activity relationship study indicated the the cis configuration and hydroxyl group in combretastatin A-4 are crucial to the antifungal effect. Molecular modeling indicated the binding sites of combretastatin A-4 and carbendazim on fungal tubulin are totally different. The bioactivity of combretastatin A-4 and its derivatives against carbendazim-resistant strains was demonstrated in this study. The results provide a new approach for fungicide discovery and fungicide resistance management.

Salvianolic acid A attenuates TNF-α- and D-GalN-induced ER stress-mediated and mitochondrial-dependent apoptosis by modulating Bax/Bcl-2 ratio and calcium release in hepatocyte LO2 cells.

Salvianolic acid (Sal A) is a water-soluble compound extracted from Radix Salvia miltiorrhiza (danshen), which has been widely used to treat acute hepatitis and hepatic damage in traditional Chinese medicine. The aim of the present study was to delineate the antiapoptotic signaling pathways involved in Sal A's hepato-protective action in hepatocyte LO2 cells and to further elucidate the mechanism by which Sal A elicits the antiapoptotic effects on hepatocytes. Here, the study showed that Sal A had antiapoptotic effects on the TNF-α/D-GalN-treated LO2 cells. Moreover, Western blotting demonstrated that the levels of p-eIF2α, ATF4, GRP78, CHOP and caspase-4 were markedly decreased in Sal A group. Additionally, the decrease of the cell mitochondrial membrane permeability and increase of ΔΨm were detected in Sal A-treated cells by high-content screening (HCS) analysis. And the levels of cleaved-caspase-9, cleaved-caspase-3, apoptosis-inducing factor (AIF), Apaf-1, and Cytc (cyto) were downregulated, while Cytc (mito) was upregulated by Sal A via Western blotting. Furthermore, the decreased levels of Bax/Bcl-2 ratio and calcium release were measured in Sal A-treated cells. In summary, Sal A attenuates TNF-α- and D-GalN-induced both ER stress and mitochondrial-dependent apoptosis by suppression of Bax/Bcl-2 ratio and prevention of calcium release, which support the notion that Sal A could be developed into a novel hepatic protectant.

Detachable strong cation exchange monolith, integrated with capillary zone electrophoresis and coupled with pH gradient elution, produces improved sensitivity and numbers of peptide identifications during bottom-up analysis of complex proteomes.

A detachable sulfonate-silica hybrid strong cation-exchange monolith was synthesized in a fused silica capillary, and used for solid phase extraction with online pH gradient elution during capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS) proteomic analysis. Tryptic digests were prepared in 50 mM formic acid and loaded onto the strong cation-exchange monolith. Fractions were eluted using a series of buffers with lower concentration but higher pH values than the 50 mM formic acid background electrolyte. This combination of elution and background electrolytes results in both sample stacking and formation of a dynamic pH junction and allows use of relatively large elution buffer volumes while maintaining reasonable peak efficiency and resolution. A series of five pH bumps were applied to elute E. coli tryptic peptides from the monolith, followed by analysis using CZE coupled to an LTQ-Orbitrap Velos mass spectrometer; 799 protein groups and 3381 peptides were identified from 50 ng of the digest in a 2.5 h analysis, which approaches the identification rate for this organism that was obtained with an Orbitrap Fusion. We attribute the improved numbers of peptide and protein identifications to the efficient fractionation by the online pH gradient elution, which decreased the complexity of the sample in each elution step and improved the signal intensity of low abundance peptides. We also performed a comparative analysis using a nanoACQUITY UltraPerformance LCH system. Similar numbers of protein and peptide identifications were produced by the two methods. Protein identifications showed significant overlap between the two methods, whereas peptide identifications were complementary.

[Study on detoxication of euphorbia pekinensis radix processed with vinegar on rat small intestinal crypt epithelial cells IEC-6].

OBJECTIVE: To compare the difference of Euphorbia Pekinensis Radix before and after being processed with vinegar in the toxicity on rat small intestinal crypt epithelial cells IEC-6, and make a preliminary study on the mechanism of detoxication of Euphorbia Pekinensis Radix processed with vinegar. METHOD: With rat small intestinal crypt epithelial cells IEC-6 as the study object, the MTT method was adopted to detect the effect of Euphorbia Pekinensis Radix before and after being processed with vinegar on IEC-6 cell activity. The morphology of cells were observed by the inverted microscope. The down-regulated mitochondrial apoptosis pathway of enterocytes caused by the vinegar processing was analyzed by using the high content screening. RESULT: Compared with the negative control group, the proliferation inhibition experiment showed that Euphorbia Pekinensis Radix showed a relatively high intestinal cell toxicity (P < 0.01). The results of HCS analysis showed that Euphorbia Pekinensis Radix could significantly reduce the cell nucleus Hoechst fluorescence intensity and mitochondria membrane (P < 0.05, P < 0.01), and increase Annexin V-FITC and PI fluorescence intensity and membrane permeability (P < 0.01, P < 0.01, P < 0.01). After being processed with vinegar, compared with Euphorbia Pekinensis Radix groups with different doses, Euphorbia Pekinensis Radix processed with vinegar could significantly decrease the cell proliferation inhibition effect on enterocytes, increase the cell nuclear Hoechst fluorescence intensity and mitochondria membrane (P < 0.05, P < 0.05), and decrease Annexin V-FITC and PI fluorescence intensity and membrane permeability (P < 0.01, P < 0.01, P < 0.05), and showed a certain dose-effect relationship. CONCLUSION: The vinegar processing can further reduce the toxicity of Euphorbia Pekinensis Radix on enterocytes. Its possible mechanism can decrease the effect of Euphorbia Pekinensis Radix on the permeability of IEC-6 cell membrane, so as to provide a basis for further explanation of the detoxication mechanism of Euphorbia Pekinensis Radix processed with vinegar.

Processing of kansui roots stir-baked with vinegar reduces kansui-induced hepatocyte cytotoxicity by decreasing the contents of toxic terpenoids and regulating the cell apoptosis pathway.

Euphorbia kansui is a Traditional Chinese Medicine widely used for the treatment of oedema, ascites and asthma. However, its serious hepatotoxicity hinders its safe clinical application. The process of stir-baking with vinegar is regularly used to reduce the toxicity of kansui. Up till now, the exact mechanism of the reduction in hepatotoxicity of kansui stir-baked with vinegar has been poorly defined. In this study, decreased  contents of five diterpene and one triterpene in kansui (GS-1) after stir-baking with vinegar (GS-2) was investigated by UPLC-QTOF/MS. Flow cytometry and Hoechst staining were used to show that the stir-baking with vinegar process reduces kansui-induced cell apoptosis. Furthermore, the result also indicated that kansui stir-baked with vinegar protects LO2 cells from apoptosis by increasing the cell mitochondrial membrane potential (ΔΨm), decreasing the release of cytochrome c and inhibiting the activities of caspase-9 and caspase-3 as evidenced by means of high content screening (HCS), ELISA and western blotting. These results suggested that the stir-baking vinegar could reduce the hepatotoxicity of kansui by effectively decreasing the contents of toxic terpenoids and inhibiting the intrinsic pathway of hepatocyte cell apoptosis. In conclusion, the study provided significant data for promoting safer and better clinical use of this herb.

Paeoniflorin protects cells from GalN/TNF-α-induced apoptosis via ER stress and mitochondria-dependent pathways in human L02 hepatocytes.

Paeoniflorin (PF) is one of the main effective components extracted from the root of Paeonia lactiflora, which has been used clinically to treat hepatitis in traditional Chinese medicine, but the details of the underlying mechanism remain unknown. The present study was designed to investigate the mechanism of protective effect of PF on d-galactosamine (GalN) and tumor necrosis factor-α (TNF-α)-induced cell apoptosis using human L02 hepatocytes. Our results confirmed that PF could attenuate GalN/TNF-α-induced apoptotic cell death in a dose-dependent manner. The disruption of mitochondrial membrane potential and the disturbance of intracellular Ca(2+) concentration were also recovered by PF. Western blot analysis revealed that GalN/TNF-α induced the activation of a number of signature endoplasmic reticulum (ER) stress and mitochondrial markers, while PF pre-treatment had a marked dose-dependent suppression on them. Additionally, the anti-apoptotic effect of PF was further evidenced by the inhibition of caspase-3/9 activities in L02 cells. These findings suggest that PF can effectively inhibit hepatocyte apoptosis and the underlying mechanism is related to the regulating mediators in ER stress and mitochondria-dependent pathways.

[Effects of component formula of Salviae Miltiorrhizae Radix et Rhizoma and Ginseng Radix et Rhizoma on cell proliferation, apoptosis and skeleton in lung cancer A549 cells].

This study aims to optimize the most effective component formula of Salviae Miltiorrhizae Radix et Rhizoma and Ginseng Radix et Rhizoma on lung cancer A549 using the orthogonal design method, and to investigate its effects of the component formula on cell proliferation, apoptosis and cytoskeleton in lung cancer A549 cells. The orthogonal design method was introduced to optimize the most effective component formula of Salviae Miltiorrhizae Radix et Rhizoma and Ginseng Radix et Rhizoma on lung cancer A549 cells. CCK-8 assay and Real-time cell analysis were adapted to analyze the effect of component formula of Salviae Miltiorrhizae Radix et Rhizoma and Ginseng Radix et Rhizoma on A549 cells viability at different time and dose. Cell apoptosis was measured by Annexin V- FITC/PI double staining and flow cytometry. Cell skeleton protein F-actin was detected by high content screening (HCS). The optimizing component formula of Salviae Miltiorrhizae Radix et Rhizoma and Ginseng Radix et Rhizoma for total salvianolic acid, total saponins of panax ginseng and ginseng polysaccharide doses were 5, 10, 5 mg L(-1). CCK-8 assay and real-time cell analysis demonstrated that the component formula of Salviae Miltiorrhizae Radix et Rhizoma and Ginseng Radix et Rhizoma treatment could significantly decrease the A549 cell viability in both dose- and time-dependent manner compared with control group (P < 0.01). Moreover, the increase of cell apoptosis was detected by Annexin V-FITC/PI double staining and flow cytometry when cells treated with the component formula, which indicating that the component formula of Salviae Miltiorrhizae Radix et Rhizoma and Ginseng Radix et Rhizoma could induce A549 cell apoptosis in a time-dependent manner compared with control group (P < 0.01). Furthermore, compared with control group, a significant decrease in A549 cell skeleton area was found in the component formula-exposed cells in the dose-dependent manner (P < 0.01). In summary, the component formula of Salviae Miltiorrhizae Radix et Rhizoma and Ginseng Radix et Rhizoma inhibits A549 cell proliferation by inducing cell apoptosis and decreasing cell microfilament formation. All of these results will be helpful to reveal antitumor mechanism of the component formula of Salviae Miltiorrhizae Radix et Rhizoma and Ginseng Radix et Rhizoma, which provides a basis for the exploration of antitumor mechanism of the component formula on lung cancer.