High-precision tracking and positioning for monitoring Holstein cattle.

Enhanced animal welfare has emerged as a pivotal element in contemporary precision animal husbandry, with bovine monitoring constituting a significant facet of precision agriculture. The evolution of intelligent agriculture in recent years has significantly facilitated the integration of drone flight monitoring tools and innovative systems, leveraging deep learning to interpret bovine behavior. Smart drones, outfitted with monitoring systems, have evolved into viable solutions for wildlife protection and monitoring as well as animal husbandry. Nevertheless, challenges arise under actual and multifaceted ranch conditions, where scale alterations, unpredictable movements, and occlusions invariably influence the accurate tracking of unmanned aerial vehicles (UAVs). To address these challenges, this manuscript proposes a tracking algorithm based on deep learning, adhering to the Joint Detection Tracking (JDT) paradigm established by the CenterTrack algorithm. This algorithm is designed to satisfy the requirements of multi-objective tracking in intricate practical scenarios. In comparison with several preeminent tracking algorithms, the proposed Multi-Object Tracking (MOT) algorithm demonstrates superior performance in Multiple Object Tracking Accuracy (MOTA), Multiple Object Tracking Precision (MOTP), and IDF1. Additionally, it exhibits enhanced efficiency in managing Identity Switches (ID), False Positives (FP), and False Negatives (FN). This algorithm proficiently mitigates the inherent challenges of MOT in complex, livestock-dense scenarios.

YAP upregulates AMPKα1 to induce cancer cell senescence.

Yes-associated protein (YAP)-a major effector protein of the Hippo pathway- regulates cell proliferation, differentiation, apoptosis, and senescence. Amp-activated protein kinase (AMPK) is a key sensor that monitors cellular nutrient supply and energy status. Although YAP and AMPK are considered to regulate cellular senescence, it is still unclear whether AMPK is involved in YAP-regulated cellular senescence. Here, we found that YAP promoted AMPKα1 aggregation and localization around mitochondria by co-transfecting CFP-YAP and YFP-AMPKα1 plasmids. Subsequent live cell fluorescence resonance energy transfer (FRET) assay did not exhibit direct interaction between YAP and AMPKα1. FRET, Co-immunoprecipitation, and western blot experiments revealed that YAP directly bound to TEAD, enhancing the expression of AMPKα1 and p-AMPKα. Treatment with verteporfin inhibited YAP's binding to TEAD and reversed the elevated expression of AMPKα1 in the cells overexpressing CFP-YAP. Verteporfin also reduced the proportion of AMPKα1 puncta in the cells co-expressing CFP-YAP and YFP-AMPKα1. In addition, the AMPKα1 puncta were demonstrated to inhibit cell viability, autophagy, and proliferation, and ultimately promote cell senescence. In conclusion, YAP binds to TEAD to upregulate AMPKα1 and promotes the formation of AMPKα1 puncta around mitochondria under the condition of co-expression of CFP-YAP and YFP-AMPKα1, in which AMPKα1 puncta lead to cellular senescence.

Regorafenib induces Bim-mediated intrinsic apoptosis by blocking AKT-mediated FOXO3a nuclear export.

Regorafenib (REGO) is a synthetic oral multi-kinase inhibitor with potent antitumor activity. In this study, we investigate the molecular mechanisms by which REGO induces apoptosis. REGO induced cytotoxicity, inhibited the proliferation and migration ability of cells, and induced nuclear condensation, and reactive oxygen species (ROS)-dependent apoptosis in cancer cells. REGO downregulated PI3K and p-AKT level, and prevented FOXO3a nuclear export. Most importantly, AKT agonist (SC79) not only inhibited REGO-induced FOXO3a nuclear localization and apoptosis but also restored the proliferation and migration ability of cancer cells, further demonstrating that REGO prevented FOXO3a nuclear export by deactivating PI3K/AKT. REGO treatment promotes Bim expression via the FOXO3a nuclear localization pathway following PI3K/AKT inactivation. REGO induced Bim upregulation and translocation into mitochondria as well as Bim-mediated Bax translocation into mitochondria. Fluorescence resonance energy transfer (FRET) analysis showed that REGO enhanced the binding of Bim to Bak/Bax. Knockdown of Bim, Bak and Bax respectively almost completely inhibited REGO-induced apoptosis, demonstrating the key role of Bim by directly activating Bax/Bak. Knockdown of Bax but not Bak inhibited REGO-induced Drp1 oligomerization in mitochondria. In conclusion, our data demonstrate that REGO promotes apoptosis via the PI3K/AKT/FOXO3a/Bim-mediated intrinsic pathway.

Sirt3 activates autophagy to prevent DOX-induced senescence by inactivating PI3K/AKT/mTOR pathway in A549 cells.

Sirtuin 3 (Sirt3), a mitochondrial deacetylase, regulates mitochondrial redox homeostasis and autophagy and is involved in physiological and pathological processes such as aging, cellular metabolism, and tumorigenesis. We here investigate how Sirt3 regulates doxorubicin (DOX)-induced senescence in lung cancer A549 cells. Sirt3 greatly reduced DOX-induced upregulation of senescence marker proteins p53, p16, p21 and SA-ß-Gal activity as well as ROS levels. Notably, Sirt3 reversed DOX-induced autophagic flux blockage, as shown by increased p62 degradation and LC3II/LC3I ratio. Importantly, the autophagy inhibitors 3-methyladenine (3-MA) and chloroquine (CQ) partially abolished the antioxidant stress and antiaging effects of Sirt3, while the autophagy activator rapamycin (Rap) potentiated these effects of Sirt3, demonstrating that autophagy mediates the anti-aging effects of Sirt3. Additionally, Sirt3 inhibited the DOX-induced activation of the phosphatidylinositol-3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway, which in turn activated autophagy. The PI3K inhibitor LY294002 promoted the antioxidant stress and antiaging effects of Sirt3, while the AKT activator SC-79 reversed these effects of Sirt3. Taken together, Sirt3 counteracts DOX-induced senescence by improving autophagic flux.

Bad is essential for Bcl-xL-enhanced Bax shuttling between mitochondria and cytosol.

Although Bcl-xL has been shown to retrotranslocate Bax from mitochondria to cytosol, other studies have found that Bcl-xL also stabilizes the mitochondrial localization of Bax. It is still unclear what causes the difference in Bcl-xL-regulated Bax localization. Bad, a BH3-only protein with a high affinity for Bcl-xL, may play an important role in Bcl-xL-regulated Bax shuttling. Here, we found that Bcl-xL enhanced both translocalization and retrotranslocation of mitochondrial Bax, as evidenced by quantitative co-localization, western blots and fluorescence loss in photobleaching (FLIP) analyses. Notably, Bad knockdown prevented Bcl-xL-mediated Bax retrotranslocation, indicating Bad was essential for this process. Quantitative fluorescence resonance energy transfer (FRET) imaging in living cells and co-immunoprecipitation analyses showed that the interaction of Bcl-xL with Bad was stronger than that with Bax. The Bad mimetic ABT-737 dissociated Bax from Bcl-xL on isolated mitochondria, suggesting that mitochondrial Bax was directly liberated to cytosol due to Bad binding to Bcl-xL. In addition, MK-2206, an Akt inhibitor, decreased Bad phosphorylation while increasing cytosolic Bax proportion. Our data firmly demonstrate a notion that Bad binds to mitochondrial Bcl-xL to release Bax, resulting in retrotranslocation of Bax to cytosol, and that the amount of Bad involved is regulated by Akt signaling.

BAK plays a key role in A-1331852-induced apoptosis in senescent chondrocytes.

Osteoarthritis occurs when the number of senescent chondrocytes in the joints reaches an intolerable level. The purpose of our study was to explore the therapeutic effect and mechanism of action of A-1331852 in osteoarthritis. Doxorubicin and etoposide were used to induce cell senescence as determined by the cessation of cell proliferation, augmented senescence-associated beta-galactosidase (SA-ß-Gal) staining, and increased p53 expression levels. The CCK-8 cytotoxicity assay and SA-ß-Gal staining demonstrated that Bcl-xL inhibitors could selectively remove senescent chondrocytes without damaging healthy chondrocytes. A-1331852 induced caspase-dependent death of senescent chondrocytes with decreased mitochondrial membrane potential, nuclear concentration, plasma membrane rupture, and PARP cleavage. Most importantly, A-1331852 upregulated BAK expression levels, indicating that BAK plays a key role in the A-1331852-induced apoptosis of senescent chondrocytes. Live-cell fluorescence resonance energy transfer showed that A-1331852 detached the binding of Bcl-xL to BAK and promoted the oligomerization of BAK on the mitochondrial membrane. In conclusion, this study provides the first evidence that A-1331852 selectively promotes apoptosis in senescent chondrocytes by interfering with the interaction between Bcl-xL and BAK.

Automated ExEm-spFRET Microscope.

Excitation­emission-spectral unmixing-based fluorescence resonance energy transfer (ExEm-spFRET) microscopy exhibits excellent robustness in living cells. We here develop an automatic ExEm-spFRET microscope with 3.04 s of time resolution for a quantitative FRET imaging. The user-friendly interface software has been designed to operate in two modes: administrator and user. Automatic background recognition, subtraction, and cell segmentation were integrated into the software, which enables FRET calibration or measurement in a one-click operation manner. In administrator mode, both correction factors and spectral fingerprints are only calibrated periodically for a stable system. In user mode, quantitative ExEm-spFRET imaging is directly implemented for FRET samples. We implemented quantitative ExEm-spFRET imaging for living cells expressing different tandem constructs (C80Y, C40Y, C10Y, and C4Y, respectively) and obtained consistent results for at least 3 months, demonstrating the stability of our microscope. Next, we investigated Bcl-xL-Bad interaction by using ExEm-spFRET imaging and FRET two-hybrid assay and found that the Bcl-xL-Bad complexes exist mainly in Bad-Bcl-xL trimers in healthy cells and Bad-Bcl-xL2 trimers in apoptotic cells. We also performed time-lapse FRET imaging on our system for living cells expressing Yellow Cameleon 3.6 (YC3.6) to monitor ionomycin-induced rapid extracellular Ca2+ influx with a time interval of 5 s for total 250 s.

Bim- and Bax-mediated mitochondrial pathway dominates abivertinib-induced apoptosis and ferroptosis.

Abivertinib (AC) is a novel epidermal growth factor receptor tyrosine kinase inhibitor with highly efficient antitumor activity. Here, we report the capacity of AC to induce both reactive oxygen species (ROS)-dependent apoptosis and ferroptosis in tumor cells. Our data showed that AC induced iron- and ROS-dependent cytotoxicity in MCF7, HeLa, and A549 cell lines. Flow cytometry analyses showed that AC increased ferrous ions and ROS and induced ferroptosis in MCF-7 cells. This was confirmed by the findings that AC not only decreased solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4) expression but also induced iron- and ROS-dependent aggrandized lipid ROS accumulation and plasma membrane damage. Meanwhile, AC induced nuclear condensation and increased ROS-dependent phosphatidylserine (PS) eversion, caspase-3 activation, and cleaved-PARP expression, suggesting that AC also induced ROS-dependent apoptosis. In addition, mitochondrial depletion significantly inhibited AC-induced cytotoxicity, including ferroptosis and apoptosis, indicating the key role of mitochondria in AC-induced ferroptosis and apoptosis. Moreover, knockout of Bim or Bax not only remarkably inhibited AC-induced apoptosis, but also markedly inhibited AC-triggered downregulation of SLC711 and GPX4, accumulation of lipid ROS, and damage to the plasma membrane. This suggests that Bim and Bax act upstream of SLC7A11 and GPX4 to mediate AC-induced ferroptosis. Collectively, AC induces ferroptosis and apoptosis, in which the Bim- and Bax-mediated mitochondrial pathways play a dominant role.

Optical section structured illumination-based Förster resonance energy transfer imaging.

Förster resonance energy transfer (FRET) microscopy is an important tool suitable for studying molecular interactions in living cells. Optical section structured illumination microscopy (OS-SIM), like confocal microscopy, has about 200 nm spatial resolution. In this report, we performed quantitative 3-cube FRET imaging in OS-SIM mode and widefield microscopy (WF) mode, respectively, for living cells expressing FRET constructs consisting of Cerulean (C, donor) and Venus (V, acceptor). OS-SIM images exhibited higher resolution than WF images. Four spectral crosstalk coefficients measured under OS-SIM mode are consistent with those measured under WF mode. Similarly, the system calibration factors G and k measured under OS-SIM mode were consistent with those measured under WF mode. The measured FRET efficiency (E) values of C32V and C17V as well as C5V constructs, standard FRET plasmids, in living Hela cells were EC32VOSF=0.32±0.02,EC17VOSF=0.38±0.02 , and EC5VOSF=0.45±0.03 , and the measured acceptor-to-donor concentration ratios ( Rc ) were RC32VOSF=1.07±0.03 , RC17VOSF=1.09±0.03 , and RC5VOSF=1.02±0.04 , consistent with the reported values. Collectively, our data demonstrates that OS-SIM can be integrated into FRET microscopy to build an OS-SIM-FRET with confocal microscopy-like resolution.

Bak instead of Bax plays a key role in metformin-induced apoptosis s in HCT116 cells.

Metformin (Met) exhibits anticancer ability in various cancer cell lines. This report aims to explore the exact molecular mechanism of Met-induced apoptosis in HCT116 cells, a human colorectal cancer cell line. Met-induced reactive oxygen species (ROS) increase and ROS-dependent cell death accompanied by plasma membrane blistering, mitochondrial swelling, loss of mitochondrial membrane potential, and release of cytochrome c. Western blotting analysis showed that Met upregulated Bak expression but downregulated Bax expression. Most importantly, silencing Bak instead of Bax inhibited Met-induced loss of mitochondrial membrane potential, indicating the key role of Bak in Met-induced apoptosis. Live-cell fluorescence resonance energy transfer (FRET) analysis showed that Met unlocked the binding of Mcl-1 to Bak, and enhanced the binding of Bim to Bak and subsequent Bak homo-oligomerization. Western blotting analysis showed that Met enhanced AMPK phosphorylation and Bim expression, and compound C, an inhibitor of AMPK, inhibited Met-induced Bim upregulation. Although Met increased the expression of Bcl-xL, overexpression of Bcl-xL did not prevent Met-induced apoptosis. In summary, our data demonstrate for the first time that Met promotes ROS-dependent apoptosis by regulating the Mcl-1-Bim-Bak axis.

Evaluating the inhibitory priority of Bcl-xL to Bad, tBid and Bax by using live-cell imaging assay.

Molecular regulatory network among the B cell leukemia-2 (Bcl-2) family proteins is a research hotspot on apoptosis. The inhibitory priority of anti-apoptotic Bcl-2 family proteins (such as Bcl-xL) to pro-apoptotic Bcl-2 family proteins (such as Bad, tBid and Bax) determines the outcome of their interactions. Based on over-expression model system, we here evaluate the inhibitory priority of Bcl-xL to Bad, tBid and Bax by using live-cell imaging assay on cell viability. Fluorescence images of living cells co-expressing CFP-Bcl-xL and YFP-Bad or YFP-tBid or YFP-Bax showed that Bcl-xL markedly inhibited Bad/tBid/Bax-mediated apoptosis, revealing that Bcl-xL inhibits the proapoptotic function of Bad, tBid and Bax. In the case of equimolar co-expression of Bad and CFP-Bcl-xL, the inhibition of Bcl-xL on tBid/Bax mediate-apoptosis was completely relieved. Moreover, co-expression of tBid-P2A-CFP-Bcl-xL significantly relieved the inhibition of Bcl-xL on the pro-apoptotic ability Bax, suggesting that Bcl-xL preferentially inhibits the pro-apoptotic ability of Bad over tBid, subsequently to Bax.

Measuring calibration factors by imaging a dish of cells expressing different tandem constructs plasmids.

Three-cube Förster resonance energy transfer (FRET) method is the most extensively applied approach for live-cell FRET quantification. Reliable measurements of calibration factors are crucial for quantitative FRET measurement. We here proposed a modified TA-G method (termed as mTA-G) to simultaneously obtain the FRET-sensitized quenching transition factor (G) and extinction coefficients ratio (γ) between donor and acceptor. mTA-G method includes four steps: (1) predetermining the ratio ranges of the sensitized emission of acceptor (FC ) to the donor excitation and donor channel image (IDD [(DA])) for all FRET plasmids; (2) culturing the cells which express every FRET plasmid in one dish respectively; (3) distinguishing and marking the cells expressing different FRET plasmids by detecting their FC /IDD (DA) values; (4) linearly fitting FC /IAA (DA) (acceptor excitation and acceptor channel image) to IDD (DA)/IAA (DA) for different kinds of cells. We implemented mTA-G method by imaging tandem constructs cells with different FRET efficiency cultured in one dish on different days, and obtained consistent G and γ values. mTA-G method not only circumvents switchover of different culture dishes but also keep the constant imaging conditions, exhibiting excellent robustness, and thus will expands the biological applications of quantitative FRET analysis in living cells.

Correction: Cepharanthine hydrochloride reverses the mdr1 (P-glycoprotein)-mediated esophageal squamous cell carcinoma cell cisplatin resistance through JNK and p53 signals.

[This corrects the article DOI: 10.18632/oncotarget.22676.].

Evaluation of the anti-cervical cancer effect of a prodrug ：CBZ-AAN-DOX with hypoxic cell culture and tumor-bearing zebrafish models.

BACKGROUND: Prodrugs are medications or compounds that, after administration, can be converted into pharmacologically active drugs through metabolism. Unlike conventional drugs, prodrugs have reduced adverse or unintended effects, which could become critical limitations in treatments such as chemotherapy. Previously through computer-aided drug design and chemical synthesis, we have obtained and examined a prodrug N-benzyloxycarbonyl-Ala-Asn-Doxorubicin (CBZ-AAN-DOX). CBZ-AAN-DOX is essentially Doxorubicin that is chemically-modified with tripeptides to target Legumain, a highly expressed protein in cancer cells and is involved in tumor metastasis and tumor microvessel formation. The difficulty to test the safety and efficacy of the prodrug (including the pharmacodynamic parameters of CBZ-AAN-DOX on metastasis and invasion of tumors, as well as cardiac and vascular toxicity) primarily comes from the lack of appropriate experimental models. METHODS: Human cervical cancer cell lines CaSki under hypoxic conditions were used to evaluate the cell viability by CCK-8 assay after the prodrug treatment. Western blotting method was performed for Legumain protein determination in the cell culture. Wound healing and transwell invasion assays were performed to determine the effects of the prodrug on tumor metastasis and invasion, respectively. Zebrafish models were constructed for toxicity and angiogenesis visual analysis after in vivo treatment with the prodrug. RESULTS: The CCK-8 results showed that CBZ-AAN-DOX exhibits an IC50 of 28.7 µM in 48 h on CaSki cells that had a lower cell inhibition rate than DOX 80.3 µM for 24 h. Legumain expression was significantly increased in a time-dependent manner in 48 h under hypoxia conditions. The results also showed that 13.9 µM of the prodrug significantly inhibited the migration and invasion of cells and the effects were significantly stronger than that of 41.8 µM of DOX under hypoxia conditions after 48 h. The effects of 160 µM of the prodrug on the survival rate of zebrafish after 72 h and heart-toxicity showed no obvious abnormalities. Cell metastasis and angiogenesis were also inhibited in tumor-bearing zebrafish model. CONCLUSION: The findings in this study demonstrated that CBZ-AAN-DOX is a promising chemotherapy candidate with low toxicity and high efficiency for cervical cancer. Remarkably, the hypoxic culture model together with the zebrafish model serve as a good system for the evaluation of the toxicity, targeting and impact of the prodrug on tumor invasion and metastasis.

Process of immunogenic cell death caused by disulfiram as the anti-colorectal cancer candidate.

BACKGROUND: Disulfiram (DSF), a drug widely used to control alcoholism, which has anticancer activity by inducing apoptosis in a copper (Cu)-dependent manner. Numerous evidences from mouse experiments indicated that some anti-cancer agents of chemotherapeutic drugs favor the induction of immunogenic cancer cell death (ICD) leading to tumor-specific immune responses. However, whether DSF could induce the colorectal tumor cells death and the mechanism involved in ICD regulatory remains elusive. The main objective of this study was to elucidate the effect of DSF/Cu on the apoptosis of colorectal cancer (CRC) cells and the expression of the two major ICD markers in CRC cells: calreticulin (CRT) and heat shock proteins (HSP) 70. METHODS: Firstly, the toxicity of DSF/Cu in HCT116, SW620 and HCT8 cells was assayed by MTT. Flow cytometry was utilized to detect the apoptosis effects. The effects of DSF/Cu on the expression of ICD-related molecules in tumor tissues were further verified in the CRC xenograft mouse model. RESULTS: The results showed that DSF/Cu increase apoptosis of these three cells in a dose dependent manner and significantly inhibited the proliferation at the concentration range from 0.05 to 1.6 µM. Furthermore, the expression of CRT and HSP70 on the cell surface also increased. The rate of transplanted tumors grew slowly, and the expression of CRT and HSP70 in colorectal cancer tissues was increased after treated with DSF/Cu. CONCLUSION: In conclusion, our results show that DSF/Cu exerts anti-colorectal cancer and its underlying mechanisms are associated with the enhancement of molecules expression of cell ICD. These results provide experimental evidence and theory basis of therapy for developing the DSF/Cu as the drug for CRC.

Magnolol attenuates the inflammation and enhances phagocytosis through the activation of MAPK, NF-κB signal pathways in vitro and in vivo.

Magnolol is a natural extract and the main bioactive component from Chinese medicine-Magnolia. We speculate that it's functional action might be associated with the anti-inflammatory effects of magnolol. Herein, the main purpose was to elucidate the phagocytic immune function and anti-inflammatory activities associated. The toxicity of magnolol on U937 and LO-2 cells was assayed by MTT, flow cytometry and laser scanning confocal microscope was utilized to detect the phagocytosis effect on U937 cells, C57BL/6 mice and the follow-up hematoxylin-eosin staining methods were used to evaluate its bioactivity in vivo. The results showed that magnolol had dose dependent effects on enhancement of phagocytosis ability and significantly inhibited the NO production at the concentration range from10 to 40 µM. Furthermore, Magnolol significantly reduced the gene expression and protein release of IL-1ß and TNF-α. However, the p-ERK1/2 in MAPK signaling pathway was not significantly affected by magnolol, whereas p-JNK and p-P38 were down-regulated. Magnolol also inhibited the expression of p-IκBα and p-P65 of NF-κB signaling pathways. The loss of body weight and the shorter length of colon were significantly improved in DSS-treated colitis C57BL/6 mice after the administration of magnolol. The cytokines of pro-inflammatory factors TNF-α, IL-6 and IL-1ß attenuated significantly in a concentration dependent manner. The histopathological manifestations of 5-20 mg/kg after the treatment magnolol were markedly improved in the DSS-treated mice. These findings showed that magnolol exerted an anti-inflammatory effect through immunoregulatory phagocytosis, MAPK and NF-κB signaling pathways. Our results provide experimental evidence and theory basis for research on anti-inflammatory effects for magnolol as a potentially anti-inflammatory drug candidate.

The IGF2/IGF1R/Nanog Signaling Pathway Regulates the Proliferation of Acute Myeloid Leukemia Stem Cells.

Acute myeloid leukemia is an aggressive disease characterized by clonal proliferation and differentiation into immature hematopoietic cells of dysfunctional myeloid precursors. Accumulating evidence shows that CD34+CD38- leukemia stem cells (LSCs) are responsible for drug resistance, metastasis, and relapse of leukemia. In this study, we found that Nanog, a transcription factor in stem cells, is significantly overexpressed in CD34+ populations from patients with acute myeloid leukemia and in LSCs from leukemia cell lines. Our data demonstrate that the knockdown of Nanog inhibited proliferation and induced cell cycle arrest and cell apoptosis. Moreover, Nanog silencing suppressed the leukemogenesis of LSCs in mice. In addition, we found that these functions of Nanog were regulated by the insulin-like growth factor receptor (IGF1R) signaling pathway. Nanog overexpression rescued the colony formation ability of LSCs treated with picropodophyllin (PPP), an IGF1R inhibitor. By contrast, knockdown of Nanog abolished the effects of IGF2 on the colony formation ability of these LSCs. These findings suggest that the IGF2/IGF1R/Nanog signaling pathway plays a critical role in LSC proliferation.

Structure Characterization of Honey-Processed Astragalus Polysaccharides and Its Anti-Inflammatory Activity In Vitro.

Honey-processed Astragalus is a dosage form of Radix Astragalus mixed with honey by a traditional Chinese medicine processing method which strengthens the tonic effect. Astragalus polysaccharide (APS), perform its immunomodulatory effects by relying on the tonic effect of Radix Astragalus, therefore, the improved pharmacological activity of honey-processed Astragalus polysaccharide (HAPS) might be due to structural changes during processing. The molecular weights of HAPS and APS were 1,695,788 Da, 2,047,756 Da, respectively, as determined by high performance gel filtration chromatography combined with evaporative light scattering detection (HPGFC-ELSD). The monosaccharide composition was determined by ultra-performance liquid chromatogram quadrupole time-of-flight mass spectrometry (UPLC/ESI-Q-TOF-MS) after pre-column derivatization with 1-phenyl-3-methyl-5-pyrazolone (PMP). The results showed that the essential components were mannose, glucose, xylose, arabinose, glucuronic acid and rhamnose, is molar ratios of 0.06:28.34:0.58:0.24:0.33:0.21 and 0.27:12.83:1.63:0.71:1.04:0.56, respectively. FT-IR and NMR analysis of HAPS results showed the presence of uronic acid and acetyl groups. The anti-inflammatory activities of HAPS were more effective than those of APS according to the NO contents and the expression of IFN-γ, IL-1ß, IL-22 and TNF-α in lipopolysaccharide (LPS)-induced RAW264.7 cells. This findings suggest that the anti-inflammatory and bioactivity improvement might be associated with molecular structure changes, bearing on the potential immunomodulatory action.

Cepharanthine hydrochloride reverses the mdr1 (P-glycoprotein)-mediated esophageal squamous cell carcinoma cell cisplatin resistance through JNK and p53 signals.

Esophageal squamous cell carcinoma (ESCC) is an aggressive malignancy that is often resistant to therapy. Nowadays, chemotherapy is still one of the main methods for the treatment of ESCC. However, the multidrug resistance (MDR)-mediated chemotherapy resistance is one of the leading causes of death. Exploring agents able to reverse MDR, which thereby increase the sensitivity with clinical first-line chemotherapy drugs, could significantly improve cancer treatment. Cepharanthine hydrochloride (CEH) has the ability to reverse the MDR in ESCC and the mechanism involved have not been reported. The aim of the study was to investigate the potential of CEH to sensitize chemotherapeutic drugs in ESCC and explore the underlying mechanisms by in vitro and in vivo studies. Our data demonstrated that CEH significantly inhibited ESCC cell proliferation in a dose-dependent manner, induced G2/M phase cell cycle arrest and apoptosis, and increased the sensitivity of cell lines resistant to cisplatin (cDDP). Mechanistically, CEH inhibited ESCC cell growth and induced apoptosis through activation of c-Jun, thereby inhibiting the expression of P-gp, and enhancing p21 expression via activation of the p53 signaling pathway. In this study, we observed that growth of xenograft tumors derived from ESCC cell lines in nude mice was also significantly inhibited by combination therapy. To our knowledge, we demonstrate for the first time that CEH is a potentially effective MDR reversal agent for ESCC, based on downregulation of the mRNA expression of MDR1 and P-gp. Together, these results reveal emphasize CEH putative role as a resistance reversal agent for ESCC.