Influence of elevated temperature on the species and mobility of chromium in ferrous sulfate-amended contaminated soil.

Ferrous sulfate (FeSO4) combined with acid pretreatment is usually employed to remediate contaminated soils containing Cr(VI). However, the long-term efficiency of this stabilization method is important for its sustainability. In this study, a gradient temperature-elevating exposure test was employed to investigate the stability of Cr in FeSO4-remediated soil when exposed to elevated temperatures (40 °C, 120 °C, and 500 °C), possibly caused by hot weather and/or wildfires. The results of chemical extraction and X-ray absorption near edge structure spectroscopy (XANES) showed that the Cr(VI) in contaminated soil was successfully transformed to Cr(III) after stabilization, resulting in the dramatic decrease of water-leachable Cr(VI). The stabilization efficiency was further improved under 40 °C treatment after 30 days. Subsequently, the 120 °C treatment (7 days) had relatively little effect on the Cr speciation and mobility in soils. However, even one day of 500 °C calcination resulted in the deterioration of stabilization efficiency, and the water-leachable Cr(VI) re-increased and became higher than the Chinese environmental standards (total Cr 15 mg/L, Cr(VI) 5 mg/L) for the classification of hazardous solid wastes. XANES results reflected that heating at 500 °C facilitate the formation of Cr2O3, which was mainly caused by thermal decomposition and dehydration of Cr(OH)3 in the soil. Besides, the transformation of Cr species resulted in the enhanced association of Cr with the most stable residual fraction (88.3%-91.6%) in soil. Based on chemical extraction results, it was suggested that the oxidation of Cr(III) to Cr(VI) contributed to the re-increased mobility of Cr(VI) in soil. However, the XANES results showed that almost no significant re-oxidization of Cr(III) to Cr(VI) happened after heating at 500 °C, which was probably caused by XANES linear combination fits (LCF) uncertainties. Moreover, the changes in soil properties, including a rise in pH to a slightly alkaline range and/or the decomposition of organic matter, possibly contributed to the enhanced mobility of Cr(VI) in soil. This study contributes to clarifying the mobility and transformation of Cr in contaminated soils and provides a support for the sustainable management of remediated soils.

Metabolic syndrome prediction model using Bayesian optimization and XGBoost based on traditional Chinese medicine features.

Metabolic syndrome (MetS) has a high prevalence and is prone to many complications. However, current MetS diagnostic methods require blood tests that are not conducive to self-testing, so a user-friendly and accurate method for predicting MetS is needed to facilitate early detection and treatment. In this study, a MetS prediction model based on a simple, small number of Traditional Chinese Medicine (TCM) clinical indicators and biological indicators combined with machine learning algorithms is investigated. Electronic medical record data from 2040 patients who visited outpatient clinics at Guangdong Chinese medicine hospitals from 2020 to 2021 were used to investigate the fusion of Bayesian optimization (BO) and eXtreme gradient boosting (XGBoost) in order to create a BO-XGBoost model for screening nineteen key features in three categories: individual bio-information, TCM indicators, and TCM habits that influence MetS prediction. Subsequently, the predictive diagnostic model for MetS was developed. The experimental results revealed that the model proposed in this paper achieved values of 93.35 %, 90.67 %, 80.40 %, and 0.920 for the F1, sensitivity, FRS, and AUC metrics, respectively. These values outperformed those of the seven other tested machine learning models. Finally, this study developed an intelligent prediction application for MetS based on the proposed model, which can be utilized by ordinary users to perform self-diagnosis through a web-based questionnaire, thereby accomplishing the objective of early detection and intervention for MetS.

Functional characterization of the GhNRT2.1e gene reveals its significant role in improving nitrogen use efficiency in Gossypium hirsutum.

Background: Nitrate is the primary type of nitrogen available to plants, which is absorbed and transported by nitrate transporter 2 (NRT2) at low nitrate conditions. Methods: Genome-wide identification of NRT2 genes in G. hirsutum was performed. Gene expression patterns were revealed using RNA-seq and qRT-PCR. Gene functions were characterized using overexpression in A. thaliana and silencing in G. hirsutum. Protein interactions were verified by yeast two-hybrid and luciferase complementation imaging (LCI) assays. Results: We identified 14, 14, seven, and seven NRT2 proteins in G. hirsutum, G. barbadense, G. raimondii, and G. arboreum. Most NRT2 proteins were predicted in the plasma membrane. The NRT2 genes were classified into four distinct groups through evolutionary relationships, with members of the same group similar in conserved motifs and gene structure. The promoter regions of NRT2 genes included many elements related to growth regulation, phytohormones, and abiotic stresses. Tissue expression pattern results revealed that most GhNRT2 genes were specifically expressed in roots. Under low nitrate conditions, GhNRT2 genes exhibited different expression levels, with GhNRT2.1e being the most up-regulated. Arabidopsis plants overexpressing GhNRT2.1e exhibited increased biomass, nitrogen and nitrate accumulation, nitrogen uptake and utilization efficiency, nitrogen-metabolizing enzyme activity, and amino acid content under low nitrate conditions. In addition, GhNRT2.1e-silenced plants exhibited suppressed nitrate uptake and accumulation, hampered plant growth, affected nitrogen metabolism processes, and reduced tolerance to low nitrate. The results showed that GhNRT2.1e could promote nitrate uptake and transport under low nitrate conditions, thus effectively increasing nitrogen use efficiency (NUE). We found that GhNRT2.1e interacts with GhNAR2.1 by yeast two-hybrid and LCI assays. Discussion: Our research lays the foundation to increase NUE and cultivate new cotton varieties with efficient nitrogen use.

Reproductive toxicity of microplastics in female mice and their offspring from induction of oxidative stress.

Microplastics (MPs) are an emerging pollutant that is becoming recognized as an increasingly serious environmental problem. The biological toxicity and resulting health risks of MPs have attracted much attention in the research community. While the effects of MPs on various mammalian organ systems have been described, their interactions with oocytes and the underlying mechanism of their activity within the reproductive system have remained ambiguous. Here, we discovered that oral administration of MPs to mice (40 mg/kg per day for 30 days) significantly reduced the oocyte maturation and fertilization rate, embryo development, and fertility. Ingestion of MPs significantly increased the ROS level in oocytes and embryos, leading to oxidative stress, mitochondrial dysfunction, and apoptosis. Moreover, mouse exposure to MPs caused DNA damage in oocytes, including spindle/chromosome morphology defects, and downregulation of actin and Juno expression in mouse oocytes. In addition, mice were also exposed to MPs (40 mg/kg per day) during gestation and lactation to determine trans-generational reproductive toxicity. The results showed that maternal exposure to MPs during pregnancy resulted in a decline in birth and postnatal body weight in offspring mice. Furthermore, MPs exposure of mothers markedly reduced oocyte maturation, fertilization rate, and embryonic development in their female offspring. This investigation provides new insights on the mechanism of MPs' reproductive toxicity and raises concerns for potential risks of MP pollution on the reproductive health of humans and animals.

[Single base editing system mediates site-directed mutagenesis of genes GDF9 and FecB in Ouler Tibetan sheep].

In this study, a single base editing system was used to edit the FecB and GDF9 gene to achieve a targeted site mutation from A to G and from C to T in Ouler Tibetan sheep fibroblasts, and to test its editing efficiency. Firstly, we designed and synthesized sgRNA sequences targeting FecB and GDF9 genes of Ouler Tibetan sheep, followed by connection to epi-ABEmax and epi-BE4max plasmids to construct vectors and electrotransfer into Ouler Tibetan sheep fibroblasts. Finally, Sanger sequencing was performed to identify the target point mutation of FecB and GDF9 genes positive cells. T-A cloning was used to estimate the editing efficiency of the single base editing system. We obtained gRNA targeting FecB and GDF9 genes and constructed the vector aiming at mutating single base of FecB and GDF9 genes in Ouler Tibetan sheep. The editing efficiency for the target site of FecB gene was 39.13%, whereas the editing efficiency for the target sites (G260, G721 and G1184) of GDF9 gene were 10.52%, 26.67% and 8.00%, respectively. Achieving single base mutation in FecB and GDF9 genes may facilitate improving the reproduction traits of Ouler Tibetan sheep with multifetal lambs.

Stereoscopic Differences in the Identification, Bioactivity, and Metabolism of C-20 and C-24 Epimeric Ginseng Saponins.

Ginseng, the roots and/or rhizomes of Panax spp.(Araliaceae), has been used as a popular herbal medicine in East Asia for at least two millennia. As a functional food and healthenhancing supplement, ginseng has been shown to have a wide range of pharmacological effects on cognition and blood circulation as well as antioxidant, antitumor, and anti-fatigue effects. The main active properties of ginseng are considered to be the triterpene saponins, often referred to as ginsenosides, which are the basis for their wide-ranging pharmacological effects. Four of these glycosides, including protopanaxadiol, protopanaxatriol, ocotillol, and oleanolic acid, are the most common saponins found in ginseng. Compared to other ginsenosides, the C-20 chimeric ginsenosides, including Rg3, Rh2, Rg2, Rh1, PF11, C-20, and C-24, as well as epimeric ocotillol-type saponins and their derivatives exhibit significant, steric differences in biological activity and metabolism. 20(R)-ginseng saponins, one class of important rare ginsenosides, have antitumor, antioxidative, antifatigue, neuroprotective and osteoclastogenesis inhibitory effects. However, 20(R)- ginsenosides are rare in natural products and are usually prepared from 20(S)-isomers through chemical differential isomerization and microbial transformation. The C20 configuration of 20(R)-ginseng saponins is usually determined by 13C NMR and X-ray single-crystal diffraction. There are regular differences in the chemical shift values of some of the carbons of the 20(S)- and 20(R)-epimers, including C-17, C-21, and C-22. Owing to their chemical structure and pharmacological and stereoselective properties, 20(R)-ginseng saponins have attracted a great deal of attention in recent years. Herein, the stereoscopic differences in the identification, bioactivity, and metabolism of C-20 and C-24 epimeric ginseng saponins are summarized.

Leaching of heavy metal(loid)s from historical Pb-Zn mining tailing in abandoned tailing deposit: Up-flow column and batch tests.

This study aims to investigate the water-leaching characteristics of heavy metal(loid)s (HMs) from historical Pb-Zn mine tailing of an abandoned tailing deposit in eastern China. Up-flow column and batch leaching tests were conducted at different liquid-to-solid (L/S) ratios to estimate the releases of HMs and investigate the controlling mechanisms. Calcite and silicate were the dominant minerals in the tailing and the HMs contents followed the order of Zn (2371 mg/kg) > Pb (2061 mg/kg) > Cu (109 mg/kg) > Cr (47.8 mg/kg) > As (15.9 mg/kg) > Cd (5.1 mg/kg). Moreover, considerable fractions of Pb, Zn, and Cd existed in the acid-soluble forms (41-47%). Column and batch leaching tests consistently showed that limited quantities (<0.002%) of HMs could be leached from this historical tailing. In particular, variations in column conditions (e.g., length, flow rate, and initial saturation) significantly affected the release fluxes from the columns but had a relatively limited effect on the leaching mechanisms. The estimated results of HM release suggested that the leaching process was predominantly solubility-controlled and the dissolution of Ca-bearing minerals (e.g., calcite) primarily controlled the release of HMs. The studied tailing had a limited impact on the quality of the surrounding aquatic environments because the water-leaching concentrations of HMs were generally lower than the Chinese standards for drinking water. Only for Pb, the leaching results in column tests were significantly lower than those in batch tests; whereas the results in column tests for other HMs were comparable to those in batch tests to a certain extent. Based on the column test results, the amounts of HMs potentially released from the abandoned tailing deposit (height, 10 m; footprint area, 30,000 m2; tailing dry density, 1.9 × 103 kg/m3) followed a decreasing order of Zn (4.2 × 105 kg) > Cu (2.3 × 104 kg) > Pb (1.4 × 104 kg) > Cr (2.3 × 104 kg) > Cd (1.6 × 103 kg) > As (1.2 × 103 kg) over the 75-year assessment period (corresponding to an L/S ratio of 10 L/kg with an annual precipitation of 1500 mm).

Allicin ameliorates imiquimod-induced psoriasis-like skin inflammation via disturbing the interaction of keratinocytes with IL-17A.

BACKGROUND AND PURPOSE: Psoriasis is an inflammatory skin disease of chronic recurrence mediated by the interaction between IL-17 and keratinocytes, which sustains a vicious circle of inflammation. Safe and effective natural medicine is a potential strategy for the clinical treatment of psoriasis. Given its prominent anti-proliferative and anti-inflammatory properties, we investigated the actions of allicin in improving psoriasis. EXPERIMENTAL APPROACH: Pharmacodynamic studies were carried out in mice after topical administration of allicin against psoriasis-like lesions induced by imiquimod. Skin sensitization tests were evaluated on guinea pigs. Toxicological studies and skin irritation tests were assessed by consecutive topical allicin alone on the skin of rabbits. RNA-seq probed transcriptomic changes following allicin. Western blot explored the actions of allicin on the interaction between IL-17A and keratinocytes. Changes in inflammatory factor expression were analysed by qPCR and immunohistochemistry. KEY RESULTS: Allicin significantly improved the epidermal structure by inhibiting the excessive proliferation and reduced apoptosis of keratinocytes. Furthermore, allicin reduced the secretion of inflammatory cytokines (IL-17A/F, IL-22, IL-12, and IL-20), chemokines (CXCL2, CXCL5, and CCL20), and anti-bacterial peptides (S100a8/9). Mechanistically, allicin directly inhibited the IL-17-induced TRAF6/MAPK/NF-κB and STAT3/NF-κB signalling cascades in keratinocytes, thus breaking the positive inflammatory feedback and alleviating imiquimod-induced psoriasis-like dermatitis in mice. Importantly, topical administration of allicin did not cause skin allergy, and the safety and adaptability of long-term application were verified. CONCLUSIONS AND IMPLICATIONS: Interfering with IL-17 signalling in keratinocytes with allicin is a promising strategy for treating psoriasis, given its safety and effectiveness.

Endophytic Klebsiella aerogenes HGG15 stimulates mulberry growth in hydro-fluctuation belt and the potential mechanisms as revealed by microbiome and metabolomics.

Growth promotion and stress tolerance induced by endophytes have been observed in various plants, but their effects on mulberry regularly suffering flood in the hydro-fluctuation belt are less understood. In the present study, endophytic Klebsiella aerogenes HGG15 was screened out from 28 plant growth promotion (PGP) bacteria as having superior PGP traits in vitro and in planta as well as biosafety for silkworms. K. aerogenes HGG15 could actively colonize into roots of mulberry and subsequently transferred to stems and leaves. The 16S ribosomal RNA (V3-V4 variable regions) amplicon sequencing revealed that exogenous application of K. aerogenes HGG15 altered the bacterial community structures of mulberry roots and stems. Moreover, the genus of Klebsiella was particularly enriched in inoculated mulberry roots and was positively correlated with mulberry development and soil potassium content. Untargeted metabolic profiles uncovered 201 differentially abundant metabolites (DEMs) between inoculated and control mulberry, with lipids and organo-heterocyclic compounds being particularly abundant DEMs. In addition, a high abundance of abiotic stress response factors and promotion growth stimulators such as glycerolipid, sphingolipid, indole, pyridine, and coumarin were observed in inoculated mulberry. Collectively, the knowledge gained from this study sheds light on potential strategies to enhance mulberry growth in hydro-fluctuation belt, and microbiome and metabolite analyses provide new insights into the growth promotion mechanisms used by plant-associated bacteria.

Identification of the CesA Subfamily and Functional Analysis of GhMCesA35 in Gossypium hirsutum L.

The cellulose synthase genes control the biosynthesis of cellulose in plants. Nonetheless, the gene family members of CesA have not been identified in the newly assembled genome of Gossypiumhirsutum (AD1, HEBAU_NDM8). We identified 38 CesA genes in G. hirsutum (NDM8) and found that the protein sequence of GhMCesA35 is 100% identical to CelA1 in a previous study. It is already known that CelA1 is involved in cellulose biosynthesis in vitro. However, the function of this gene in vivo has not been validated. In this study, we verified the function of GhMCesA35 in vivo based on overexpressed Arabidopsis thaliana. In addition, we found that it interacted with GhCesA7 through the yeast two-hybrid assay. This study provides new insights for studying the biological functions of CesA genes in G. hirsutum, thereby improving cotton fiber quality and yield.

Therapeutic targeting of hepatic ACSL4 ameliorates NASH in mice.

BACKGROUND AND AIMS: Globally, NAFLD is one of the most common liver disorders, with an estimated prevalence rate of more than 30% in men and 15% in women and an even higher prevalence in people with type 2 diabetes mellitus. Optimal pharmacologic therapeutic approaches for NAFLD are an urgent necessity. APPROACH AND RESULTS: In this study, we showed that compared with healthy controls, hepatic ACSL4 levels in patients with NAFLD were found to be elevated. Suppression of ACSL4 expression promoted mitochondrial respiration, thereby enhancing the capacity of hepatocytes to mediate ß-oxidation of fatty acids and to minimize lipid accumulation by up-regulating peroxisome proliferator-activated receptor coactivator-1 alpha. Moreover, we found that abemaciclib is a potent and selective ACSL4 inhibitor, and low dose of abemaciclib significantly ameliorated most of the NAFLD symptoms in multiple NAFLD mice models. CONCLUSIONS: Therefore, inhibition of ACSL4 is a potential alternative therapeutic approach for NAFLD.

Molecule mechanisms of Ganoderma lucidum treated hepatocellular carcinoma based on the transcriptional profiles and miRNA-target network.

Ganoderma lucidum has salutary effects on tumor treatment, including pancreatic cancer and hepatocellular carcinoma. However, the molecular mechanisms underlying Ganoderma lucidum therapy is obscure. In this study, the Hepa1-6-bearing C57 BL/6 mouse model was utilized to explore the therapeutic efficacy of Ganoderma lucidum extract (GLE), documenting that it could effectively inhibit tumor growth. The microRNA (miRNA) profiles of GLE-treated and untreated mice were detected, and 25 differentially expressed (DE) miRNAs were determined, including 24 up-expressed and one down-expressed miRNAs. Using the ClusterOne algorithm, 8 hub miRNAs were isolated from the established miRNA-target network. The qRT-PCR assay demonstrated that these 8 miRNAs were up-expressed in the GLE treated tumor mice. Furthermore, the mRNA profiles showed that there are 76 DE mRNAs between GLE treated and model groups. The protein-protein interaction (PPI) network shows that Cntn1, Irs1, Nfkbia, Rybp and Ywhaz playing important roles, and qRT-PCR further revealed they were down-expressed in GLE treated Hepa1-6-bearing C57 BL/6 mice. The rebuilt miRNA-target network was shown that these 5 mRNAs were regulated by mmu-mir-23a-5p, -3102-3p, -337-3p, and -467a-3p, respectively. This study suggested that these 4 interesting miRNAs were potential biomarkers for evaluation of GLE efficacy, which may down-regulate the expression of Cntn1, Irs1, Nfkbia, Rybp and Ywhaz, and mediate many signaling pathways occurring in tumor treatment.

Ochratoxin A exposure causes meiotic failure and oocyte deterioration in mice.

Ochratoxin A (OTA) is a mycotoxin produced by fungi and occurs naturally in various foodstuffs and some animal-derived products. This mycotoxin can cause deleterious effects on kidney, liver, central nervous, and immune system. However, potential mechanisms regarding how OTA disrupts the mammalian oocyte quality have not been clearly defined. In this study, we proved that OTA weakened oocyte quality by impairing oocyte meiotic maturation. We found that female mice treated with 1 mg/kg body weight OTA by intraperitoneal (IP) injection for 7 days displayed ovarian dysfunction and decreased offspring number. We also found that OTA treatment at 7.5 µM for 16 h decreased the rate of first polar body extrusion by disrupting spindle and chromosome alignment. In addition, OTA caused oxidative stress by inducing the accumulation of reactive oxygen species and consumption of antioxidants during meiosis, consequently resulting in oocytes apoptosis. Mitochondrial damage and insufficient energy supply were also observed in OTA-pretreated oocytes, which led to the meiotic failure of oocyte. Moreover, the epigenetic modifications were also affected, showing with altered 5 mC, 5hmC, H3K9ac, and H3K9me3 levels in mice oocytes. In summary, these results showed that OTA could decrease oocyte maturation and fertility by inducing oxidative stress and epigenetic changes.

The effect of Ganoderma lucidum extract on immunological function and identify its anti-tumor immunostimulatory activity based on the biological network.

Ganoderma lucidum extract (GLE) has shown positive effects for tumor treatment. However, the molecular mechanism of GLE treatment is unknown. In this study, a Hepa1-6-bearing C57 BL/6 mouse model was established to explore the anti-tumor and immunostimulatory activity of GLE treatment. The results showed that GLE effectively inhibited tumor growth without hepatic/renal toxicity and bone marrow suppression, and might enhancing immunological function. Based on the mRNA profiles of GLE treated and untreated mice, 302 differentially expressed (DE) mRNAs were identified and 6 kernel mRNAs were identified from the established protein-protein interaction (PPI) network. Quantitative RT-PCR and western-blot analysis indicated that 6 mRNAs have had statistically significant differences between the GLE treated and untreated mice. Furthermore, four kernel pathways were isolated from the KEGG-Target network, including the Jak-STAT signaling pathway, T cell receptor signaling pathway, PI3K-Akt signaling pathway, and cytokine-cytokine receptor interaction. Western-blot and cytokine detection results demonstrated that GLE suppressed growth and proliferation of tumors by the Jak-STAT signaling pathway, T cell receptor signaling pathway and PI3K-Akt signaling pathway, but also regulated the expression levels of serum immune cytokines and improved the anti-tumor immunostimulatory activity.

Network pharmacology analysis of the anti-cancer pharmacological mechanisms of Ganoderma lucidum extract with experimental support using Hepa1-6-bearing C57 BL/6 mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Ganoderma lucidum (GL) is an oriental medical fungus, which was used to prevent and treat many diseases. Previously, the effective compounds of Ganoderma lucidum extract (GLE) were extracted from two kinds of GL, [Ganoderma lucidum (Leyss. Ex Fr.) Karst.] and [Ganoderma sinense Zhao, Xu et Zhang], which have been used for adjuvant anti-cancer clinical therapy for more than 20 years. However, its concrete active compounds and its regulation mechanisms on tumor are unclear. AIM OF THE STUDY: In this study, we aimed to identify the main active compounds from GLE and to investigate its anti-cancer mechanisms via drug-target biological network construction and prediction. MATERIALS AND METHODS: The main active compounds of GLE were identified by HPLC, EI-MS and NMR, and the compounds related targets were predicted using docking program. To investigate the functions of GL holistically, the active compounds of GL and related targets were predicted based on four public databases. Subsequently, the Identified-Compound-Target network and Predicted-Compound-Target network were constructed respectively, and they were overlapped to detect the hub potential targets in both networks. Furthermore, the qRT-PCR and western-blot assays were used to validate the expression levels of target genes in GLE treated Hepa1-6-bearing C57 BL/6 mice. RESULTS: In our work, 12 active compounds of GLE were identified, including Ganoderic acid A, Ganoderenic acid A, Ganoderic acid B, Ganoderic acid H, Ganoderic acid C2, Ganoderenic acid D, Ganoderic acid D, Ganoderenic acid G, Ganoderic acid Y, Kaemferol, Genistein and Ergosterol. Using the docking program, 20 targets were mapped to 12 compounds of GLE. Furthermore, 122 effective active compounds of GL and 116 targets were holistically predicted using public databases. Compare with the Identified-Compound-Target network and Predicted-Compound-Target network, 6 hub targets were screened, including AR, CHRM2, ESR1, NR3C1, NR3C2 and PGR, which was considered as potential markers and might play important roles in the process of GLE treatment. GLE effectively inhibited tumor growth in Hepa1-6-bearing C57 BL/6 mice. Finally, consistent with the results of qRT-PCR data, the results of western-blot assay demonstrated the expression levels of PGR and ESR1 were up-regulated, as well as the expression levels of NR3C2 and AR were down-regulated, while the change of NR3C1 and CHRM2 had no statistical significance. CONCLUSIONS: The results indicated that these 4 hub target genes, including NR3C2, AR, ESR1 and PGR, might act as potential markers to evaluate the curative effect of GLE treatment in tumor. And, the combined data provide preliminary study of the pharmacological mechanisms of GLE, which may be a promising potential therapeutic and chemopreventative candidate for anti-cancer.

Ophiopogonin B induces apoptosis, mitotic catastrophe and autophagy in A549 cells.

Ophiopogonin B (OP-B), a saponin compound isolated from Radix Ophiopogon japonicus, was verified to inhibit cell proliferation in numerous non-small cell lung cancer (NSCLC) cells in our previous study. However, the precise mechanisms of action have remained unclear. In the present study, we mainly investigated the effects of OP-B on adenocarcinoma A549 cells to further elaborate the underlying mechanisms of OP-B in different NSCLC cell lines. Detection by high content screening (HCS) and TUNEL assay verified that OP-B induced apoptosis in this cell line, while detection of Caspase-3, Bcl-2 and Bax showed that OP-B induced cell death was caspase and mitochondrial independent. Further experiments showed that OP-B induced cell cycle arrest in the S and G2/M phases by inhibiting the expression of Myt1 and phosphorylation of Histone H3 (Ser10), which resulted in mitotic catastrophe in the cells. Transmission electron microscopy (TEM) observation of cell micro-morphology combined with detection of Atgs by western blot analysis showed that OP-B induced autophagy in this cell line. Autophagy inhibition by the lysosome inhibitor CQ or Beclin1-siRNA knockdown both attenuated cell viability, demonstrated that autophagy also being the vital reason resulted in cell death. More importantly, the xenograft model using A549 cells provided further evidence of the inhibition of OP-B on tumor proliferation. Immunohistochemistry detection of LC3 and Tunel assay both verified that high dose of OP-B (75 mg/kg) induced autophagy and apoptosis in vivo, and western blot detection of p-Histone H3 (Ser10), Survivin and XIAP further indicated the molecular mechanism of OP-B in vivo. As our findings revealed, multiple types of cell death overlapped in OP-B treated A549 cells, it displayed multitarget characteristics of the compounds extracted from the Chinese herbal, which may be used as candidate anticancer medicine in clinic.

Platycodin-D Induced Autophagy in Non-Small Cell Lung Cancer Cells via PI3K/Akt/mTOR and MAPK Signaling Pathways.

Platycodin-D (PD) is an effective triterpene saponin extracted from the root of Platycodon grandiflorum which has been used clinically to treat pulmonary diseases in traditional Chinese medicine. Recently, it has been reported that PD has anti-tumor effects in various cancer models through the induction of apoptosis. However, whether PD induces autophagy in both cell lines and its molecular mechanisms have not been elucidated. Here, our present study confirmed that PD induced autophagy in both NCI-H460 and A549 cells via up-regulating the expression levels of Atg-3, Atg-7 and Beclin-1. Meanwhile, PD contributed to the up-regulation of LC3-II at both protein and mRNA levels. Further detection of the PI3K/Akt/mTOR signaling pathway compared to LY294002 (PI3K kinase inhibitor), RAP (mTOR kinase inhibitor) and insulin (an activator of PI3K/Akt/mTOR signaling pathway) showed that PD induced autophagy through inhibiting the pathway at p-Akt (Ser473), p-p70S6K (Thr389) and p-4EBP1 (Thr37/46) in both cell lines. Moreover, the examination of MAPK signaling pathway showed that PD treatment increased the phosphorylation of JNK and p38 MAPK, while decreased the phosphorylation of Erk1/2 in both cell lines. Additionally, the effects assessed with a panel of pharmacologic inhibitors, including U0126 (Erk1/2 kinase inhibitor), SP600125 (JNK kinase inhibitor) and SB203580 (p38 MAPK kinase inhibitor) suggested that the activation of JNK and p38 MAPK participated in PD-induced autophagy. Taken together, these findings suggested that PD induced autophagy in NCI-H460 and A549 cells through inhibiting PI3K/Akt/mTOR signaling pathway and activating JNK and p38 MAPK signaling pathways. Therefore, PD may be an alternative compound for NSCLC therapy.

Glycyrrhetinic acid induces G1­phase cell cycle arrest in human non­small cell lung cancer cells through endoplasmic reticulum stress pathway.

Glycyrrhetinic acid (GA) is a natural compound extracted from liquorice, which is often used in traditional Chinese medicine. The purpose of the present study was to investigate the antitumor effect of GA in human non­small cell lung cancer (NSCLC), and its underlying mechanisms in vitro. We have shown that GA suppressed the proliferation of A549 and NCI­H460 cells. Flow cytometric analysis showed that GA arrested cell cycle in G0/G1 phase without inducing apoptosis. Western blot analysis indicated that GA mediated G1­phase cell cycle arrest by upregulation of cyclin­dependent kinase inhibitors (CKIs) (p18, p16, p27 and p21) and inhibition of cyclins (cyclin­D1, ­D3 and ­E) and cyclin­dependent kinases (CDKs) (CDK4, 6 and 2). GA also maintained pRb phosphorylation status, and inhibited E2F transcription factor 1 (E2F­1) in both cell lines. GA upregulated the unfolded proteins, Bip, PERK and ERP72. Accumulation of unfolded proteins in the endoplasmic reticulum (ER) triggered the unfolded protein response (UPR), which could be the mechanism by which GA inhibited cell proliferation in NSCLC cells. GA then coordinated the induction of ER chaperones, which decreased protein synthesis and induced cell cycle arrest in the G1 phase. This study provides experimental evidence to support the development of GA as a chemotherapeutic agent for NSCLC.

[Study on synergistic effect of sodium cantharidinate combined with chemotherapeutic drugs on hepatic carcinoma and its effective mechanism].

OBJECTIVE: To study the synergistic effect on hepatoma cell(SMMC-7721) and the reduction killing effect on normal liver cells(LO-2) treated with sodium cantharidinate (SCA) in combination with fluorouracil(5-FU) or cisplatin(DDP) as well as the related mechanism. METHODS: MTT assay was used to select the best ratio of SCA with 5-FU or SCA with DDP which had less toxicity on LO-2 cell line and had synergistic effect on SMMC-7721 cell line; Flow cytometry assay was used to analyze the apoptosis-induction of the different ratio of drugs on both cell lines; Hoechst-33258 fluorescent staining assay was used to observe the nuclear morphological changes of cells; Immunoblotting assay was used to analyze the Ras/Raf/ERK1/2 signaling pathway and the apoptosis related signaling pathway in both cell lines. RESULTS: MTI assay indicated that the proliferation inhibition of SCA,5-FU and DDP on SMMC-7721 cell line was in a time-and dose-dependent manner respectively. Among them, SCA had a more significant inhibition on SMMC-7721 cell line than on LO-2 after 12 h or 24 h treatment (P <0. 01). Moreover, after a treatment of 48 h,the ratio of 2. 5 µg/mL SCA and 2 µg/mL DDP showed a more significant inhibition on SMMC-7721 cell line than on LO-2 cell line,which was then be considered as the optimal concentration ratio for the following experiment. Co-treatment of SCA (2. 5 µg/mL) with DDP (2 µg/mL) induced a more significant apoptosis on SMMC-7721 cell line compared with single treatment with SCA (2. 5 µg/mL) or DDP (2 µg/mL) respectively (P < 0. 01). After a 48 h treatment of the optimal ratio of drugs, the significant morphological apoptotic characteristics were observed both under inverted microscope and by Hoechst-33258 fluorescent staining assay in both cell lines. The results of Western blot assay showed that this ratio of drugs could significantly increase the protein expression of Bax,P53 and P21 and decreased the expression of BCL-2, Casepase-3, p-Erk, p-Ras and p-c-Raf in SMMC-7721 cells. Meanwhile,the effect on the proteins mentioned above was lesser in LO-2 cells. CONCLUSION: These results indicates that 2. 5 µg/mL SCA + 2 µg/mL DDP showed a higher inhibition on the hepatic carcinoma cells and a relatively lower cytotoxicity on normal liver cells. The major anti-cancer mechanism is related with the inhibition on Erk signaling pathway and the induction of apoptosis through the mitochondrial pathway.