Ultra-Small Copper-Based Multienzyme-Like Nanoparticles Protect Against Hepatic Ischemia-Reperfusion Injury Through Scavenging Reactive Oxygen Species in Mice.

Hepatic ischemia-reperfusion injury (IRI) is a severe complication that occurs in the process of liver transplantation, hepatectomy, and other end-stage liver disease surgery, often resulting in the failure of surgery operation and even patient death. Currently, there is no effective way to prevent hepatic IRI clinically. Here, it is reported that the ultra-small copper-based multienzyme-like nanoparticles with catalase-like (CAT-like) and superoxide dismutase-like (SOD-like) catalytic activities significantly scavenge the surge-generated endogenous reactive oxygen species (ROS) and effectively protects hepatic IRI. Density functional theory calculations confirm that the nanoparticles efficiently scavenge ROS through their synergistic effects of the ultra-small copper SOD-like activity and manganese dioxides CAT-like activity. Furthermore, the results show that the biocompatible CMP NPs significantly protected hepatocytes from IRI in vitro and in vivo. Importantly, their therapeutic effect is much stronger than that of N-acetylcysteamine acid (NAC), an FDA-approved antioxidative drug. Finally, it is demonstrated that the protective effects of CMP NPs on hepatic IRI are related to suppressing inflammation and hepatocytic apoptosis and maintaining endothelial functions through scavenging ROS in liver tissues. The study can provide insight into the development of next-generation nanomedicines for scavenging ROS.

Metal-ligand cross-link strategy engineered iron-doped dopamine-based superstructure as peroxidase-like nanozymes for detection of glucose.

Nanozymes are nanomaterials with mimetic enzyme properties and the related research has attracted much attention. It is of great value to develop methods to construct nanozymes and to study their application in bioanalysis. Herein, the metal-ligand cross-linking strategy was developed to fabricate superstructure nanozymes. This strategy takes advantage of being easy to operate, adjustable, cheap, and universal. The fabricated superstructure nanozymes possess efficient peroxidase-like catalytic activity. The enzyme reaction kinetic tests demonstrated that for TMB and H2O2, the Km is 0.229 and 1.308 mM, respectively. Furthermore, these superstructure nanozymes are applied to highly efficient and sensitive detection of glucose. The linear range for detecting glucose is 20-2000 µM, and the limit of detection is 17.5 µM. Furthermore, mechanistic research illustrated that this integrated system oxidizes glucose to produce hydrogen peroxide and further catalyzes the production of ·OH and O2·-, which results in a chromogenic reaction of oxidized TMB for the detection of glucose. This work could not only contribute to the development of efficient nanozymes but also inspire research in the highly sensitive detection of other biomarkers.

Dual-Functional Capping Agent-Mediated Transformation of Silver Nanotriangles to Silver Nanoclusters for Dual-Mode Biosensing.

The localized surface plasmon resonance (LSPR) property, depending on the structure (morphology and assembly) of nanoparticles, is very sensitive to the environmental fluctuation. Retaining the colorimetric effect derived from the LSPR property while introducing new optical properties (such as fluorescence) that provide supplementary information is an effective means to improve the controllability in structures and reproducibility in optical properties. DNA as a green and low-cost etching agent has been demonstrated to effectively control the morphology and optical properties (the blue shift of the LSPR peak) of the plasmonic nanoparticles. Herein, taking silver nanotriangles (AgNTs) as a proof of concept, we report a novel strategy to induce precisely tunable LSPR and fluorescence-composited dual-mode signals by using mono-DNA first as an etching agent for etching the morphology of AgNTs and later as a template for synthesizing fluorescent silver nanoclusters (AgNCs). In addition, common templates for synthesizing AgNCs, such as l-glutathione and bovine serum albumin, were demonstrated to have the capability to serve as etching agents. More importantly, these biomolecules as dual-functional capping agents (etching agents and templates) follow the size-dependent rule: as the size of the thiolated biomolecule increases, the blue shift of the LSPR peak increases; at the same time, the fluorescence intensity increases. The enzyme that can change the molecular weight (size) of the biomolecular substrates (DNA, peptides, and proteins) through an enzymatic cleavage reaction was explored to regulate the LSPR and fluorescent properties of the resulting nanoparticles (by etching of AgNTs and synthesis of AgNCs), achieving excellent performance in detection of cancer-related proteases. This study can be expanded to other biopolymers to impact both fundamental nanoscience and applications and provide powerful new tools for bioanalytical biosensors and nanomedicine.

[Review of classical prescriptions in treatment of ulcerative colitis].

Ulcerative colitis(UC) is a continuous inflammatory bowel disease with the main clinical manifestations of abdominal pain, diarrhea, and mucous bloody stools, mainly attacking the colorectal mucosa and submucosa. It is characterized by high recurrence rate, difficult cure, and clustering and regional occurrence. Chinese medicinal prescriptions for the treatment of UC have good therapeutic effect, multi-target regulation, slight toxicity, and no obvious side effects. In particular, the classical prescriptions highlight the characteristics and advantages of traditional Chinese medicine theory and have attracted much attention in recent years. To enable researchers to timely and comprehensively understand the classical prescriptions in the treatment of UC, we reviewed the studies about the pharmacodynamic material basis, quality control, action mechanism, and clinical application of relevant classical prescriptions. We first introduced the latest research progress in the active components such as alkaloids, polysaccharides, saponins, and flavonoids in relevant classical prescriptions. Then, we reviewed the latest research achievements on the quality control of classical prescriptions for the treatment of UC by gas chromatography, liquid chromatography, mass spectrometry, liquid chromatography-mass spectrometry and the like. Further, we summarized the research advances in the mechanisms of relevant prescriptions in the treatment of UC based on network pharmacology, molecular docking, integrated pharmacology platform, and animal experiments. Finally, we generalized the clinical application of the classical prescriptions for clearing heat and removing dampness, mildly regulating cold and heat, soothing liver and regulating spleen, strengthening spleen and invigorating Qi, and tonifying spleen and stomach. By systematic summary of the research progress in relevant classical prescriptions, we hope to promote the application and development of such prescriptions in UC treatment.

Integral Multielement Signals by DNA-Programmed UCNP-AuNP Nanosatellite Assemblies for Ultrasensitive ICP-MS Detection of Exosomal Proteins and Cancer Identification.

Exosomes are expected to be used as cancer biomarkers because they carry a variety of cancer-related proteins inherited from parental cells. However, it is still challenging to develop a sensitive, robust, and high-throughput technique for simultaneous detection of exosomal proteins. Herein, three aptamers specific to cancer-associated proteins (CD63, EpCAM, and HER2) are selected to connect gold nanoparticles (AuNPs) as core with three different elements (Y, Eu, and Tb) doped up-conversion nanoparticles (UCNPs) as satellites, thereby forming three nanosatellite assemblies. The presence of exosomes causes specific aptamers to recognize surface proteins and release the corresponding UCNPs, which can be simultaneously detected by inductively coupled plasma-mass spectrometry (ICP-MS). It is worth noting that rare earth elements are scarcely present in living systems, which minimize the background for ICP-MS detection and exclude potential interferences from the coexisting species. Using this method, we are able to simultaneously detect three exosomal proteins within 40 min, and the limit of detection for exosome is 4.7 × 103 particles/mL. The exosomes from seven different cell lines (L-02, HepG2, GES-1, MGC803, AGS, HeLa, and MCF-7) can be distinguished with 100% accuracy by linear discriminant analysis. In addition, this analytical strategy is successfully used to detect exosomes in clinical samples to distinguish stomach cancer patients from healthy individuals. These results suggest that this sensitive and high-throughput analytical strategy based on ICP-MS has the potential to play an important role in the detection of multiple exosomal proteins and the identification of early cancer.

Precisely Tuning LSPR Property via "Peptide-Encoded" Morphological Evolution of Gold Nanorods for Quantitative Visualization of Enzyme Activity.

Longitudinal surface plasmon resonance (LSPR)-based optical signals possess unique advantages in biomolecular sensing and detection which can be attributed to their ultrahigh sensitivity and signal-to-noise ratio. However, the lack of effective strategies for morphological control of gold nanorods (GNRs) complicates the precise tuning of their LSPR property. Herein, a "peptide-encoded" strategy was first developed to precisely control the morphologies of GNRs via overgrowth of GNR seeds in the presence of thiol-containing peptides. Significantly, the "peptide-encoded" GNRs exhibit a tunable LSPR peak ranging from 685 to 877 nm by altering the amount of peptide. A few obvious colorimetric changes were accompanied from pink to purple and even to blue. Other parameters, e.g., pH, temperature, and Ag+ concentration, could also be utilized to regulate the morphologies of the "peptide-encoded" GNRs. The ultrasensitive detection of tumor-related protease activities based on LSPR peak shifts was further successfully performed without the need for labeling or instrumental aid, achieving a limit of detection of 60 fM. It is much lower than traditional absorption-based analysis (1 nM) and enzyme-linked immunosorbent assay (ELISA) method (1 pM), indicating the great potential of this peptide-encoded strategy in the application of ultrasensitive biomarker assay and clinical diagnosis.

Capping Ligand Size-Dependent LSPR Property Based on DNA Nanostructure-Mediated Morphological Evolution of Gold Nanorods for Ultrasensitive Visualization of Target DNA.

Systematically tuning the structures and properties of noble-metal nanoparticles through biomolecule-mediated overgrowth is of significant importance for their applications in biosensing and imaging. Herein thiolated biomolecules with different concentrations and sizes (molecular weight and spatial structure) were used as a class of capping ligands to control the longitudinal surface plasmon resonance (LSPR) property of gold nanorods (GNRs). The LSPR peaks were red-shifted by increasing the capping agent concentration. The size effect could be divided to two aspects: (1) When the ligands are small molecules, the LSPR peak is blue-shifted as the size of the capping ligand increases. (2) When the ligands are macromolecular proteins, the LSPR property is similar to that of the overgrown nanoparticle (Au@gap@GNR) without thiolated biomolecules as capping agents. Interestingly, thiol-free and nonhomooligomeric DNA strands as capping agents present a similar influence in shaping the overgrowth of GNRs by varying their concentrations and sizes. In addition, the size effect of a DNA nanostructure was used to construct a ΔλLSPR-based catalytic nucleic acid biosensor using a DNA dendritic nanostructure as a capping agent combined with LSPR signals generated from the Au@gap@GNRs with morphological evolution. More importantly, the ΔλLSPR-based biosensor possesses three advantages in nucleic acid biosensing: (1) It is completely label- and wash-free, (2) it has an ultrahigh sensitivity and signal-to-noise ratio, and (3) it can be visualized without any instrumental aid, indicating a significant potential for ultrasensitive biosensing.

A simple enzyme-assisted cascade amplification strategy for ultrasensitive and label-free detection of DNA.

A simple fluorescence biosensor is developed based on the enzyme-assisted cascade amplification strategy. The amplification system consists of a hairpin-structure DNA (H-DNA) and exonuclease III. The target DNA can hybridize with the H-DNA and initiate exonuclease III-assisted target recycling amplification to generate abundant G-rich DNA (G-DNA). One region of G-DNA is designed to possess the same sequence as target DNA. Thus, the G-DNA can also hybridize with H-DNA and initiate the digestion of H-DNA. The cascade strategy in this amplification system causes the concentration of G-DNA to grow exponentially. The fluorescence intensity of N-methylmesoporphyrin IX (NMM) is highly enhanced due to the formation of G-quadruplex configuration. Under optimal conditions, the cascade system could achieve an admirable sensitivity with a detection limit of 52 fM for HIV DNA, and guarantees a satisfactory specificity. Moreover, the cascade system could be implemented for other target DNA detections by substituting the recognition region of the H-DNA. In this way, a detection limit of 65 fM for HBV DNA could be achieved by the cascade system. The target DNA analysis in a real serum sample further indicates that this biosensor has potential for future application in clinical diagnosis. Graphical abstract A simple and label-free cascade amplification strategy is developed by exploiting hairpin DNA and EXO III for sensitive DNA detection.

Smart DNA Machine for Carcinoembryonic Antigen Detection by Exonuclease III-Assisted Target Recycling and DNA Walker Cascade Amplification.

A synthetic DNA machine performs quasi-mechanical movements in response to external intervention, suggesting the promise of constructing sensitive and specific biosensors. Herein, a smart DNA walker biosensor for label-free detection of carcinoembryonic antigen (CEA) is developed for the first time by a novel cascade amplification strategy of exonuclease (Exo) III-assisted target recycling amplification (ERA) and DNA walker. ERA as the first stage of amplification generates the walker DNA, while the autonomous traveling of the walker DNA on the substrate-modified silica microspheres as the second stage of amplification produces an ultrasensitive fluorescent signal with the help of N-methylmesoporphyrin IX (NMM). The DNA machine as a biosensor could be applied for transducing and quantifying signals from isothermal molecular amplifications, avoiding the complicated reporter elements and thermal cycling. The present biosensor achieves a detection limit of 1.2 pg·mL-1 within a linear range of 10 pg·mL-1 to 100 ng·mL-1 for CEA, along with a favorable specificity. The practical applicability of the biosensor is demonstrated by the detection of CEA in human serum with satisfactory results; thus, it shows great potential in clinical diagnosis.

A sensitive aptasensor based on molybdenum carbide nanotubes and label-free aptamer for detection of bisphenol A.

To specifically and sensitively identify bisphenol A (BPA) with a simple and rapid method is very important for food safety. Using an anti-BPA aptamer and Mo2C nanotubes, we developed a label-free and low-background signal biosensor for BPA detection. The anti-BPA aptamer drastically increased the fluorescence signal of N-methylmesoporphyrin IX under an assistance of Help-DNA. Additionally, BPA can interact with the anti-BPA aptamer and switch its conformation to prevent the formation of a G-quadruplex, resulting in fluorescence quenching. Simultaneously, Mo2C nanotubes can reduce the background signals due to the adsorption of Help-DNA on their surface. This method shows a linear range of 2-20 nM with a detection limit of 2 nM for detecting BPA. This label-free BPA aptasensor with low background signal is inexpensive, easy to use, and can be applied to determine BPA in real water samples. Graphical Abstract A low-background and label-free biosensor was designed based on Mo2C nanotubes and aptamer for BPA detection.

Inhibition of CYP1A1 expression enhances diabetic wound healing by modulating inflammation and oxidative stress in a rat model.

OBJECTIVE: Wound therapies utilizing gene delivery to the skin offer considerable promise owing to their localized treatment benefits and straightforward application. This study investigated the impact of skin electroporation of CYP1A1 shRNA lentiviral particles on diabetic wound healing in a streptozotocin (STZ)-induced rat model. METHODS: Male Sprague Dawley (SD) rats were made diabetic by injecting STZ and subsequently creating foot skin wounds. The rats were randomly divided into four groups: normal, diabetic foot ulcers (DFU), DFU + control shRNA (electroporation of control shRNA lentiviral particles), and DFU + CYP1A1 shRNA (electroporation of CYP1A1 shRNA lentiviral particles). Wound healing progress was monitored at multiple time points (0, 1, 3, 5, 7, 10, 14 days). On day 14, wound tissue specimens were collected for histological examination. Wound samples collected at days 7 and 14 were used for gene expression analysis via qRT-PCR, assessment of CYP1A1 protein levels using western blotting, and evaluation of oxidative stress markers. RESULTS: Treatment with CYP1A1 shRNA significantly enhanced diabetic wound healing rates compared to untreated controls over the observation period. Histological analysis revealed improved wound characteristics in the CYP1A1 shRNA-treated group, including enhanced epithelial regeneration, reduced inflammation, and increased collagen deposition, indicative of improved tissue repair. Furthermore, suppression of CYP1A1 corresponded with decreased expression levels of pro-inflammatory cytokines (interleukin-1ß, tumor necrosis factor-α, and interleukin-6) and diminished oxidative stress markers (malondialdehyde, superoxide dismutase) within wound tissues. CONCLUSION: Targeted suppression of CYP1A1 represents a promising therapeutic strategy to enhance diabetic wound healing by modulating inflammation and oxidative stress.

Mechanism interpretation of Guhan Yangshengjing for protection against Alzheimer's disease by network pharmacology and molecular docking.

ETHNOPHARMACOLOGICAL RELEVANCE: Guhan Yangshengjing (GHYSJ) is an effective prescription for delaying progression of Alzheimer's disease (AD) based on the ancient Chinese medical classics excavated from Mawangdui Han Tomb. Comprising a combination of eleven traditional Chinese herbs, the precise protective mechanism through which GHYSJ acts on AD progression remains unclear and has significant implications for the development of new drugs to treat AD. AIM OF THE STUDY: To investigate the mechanism of GHYSJ in the treatment of AD through network pharmacology and validate the results through in vitro experiments. MATERIALS AND METHODS: Chemical composition-target-pathway network and protein-protein interaction network were constructed by network pharmacology to predict the potential targets of GHYSJ for the treatment of AD. The interaction relationship between active ingredients and targets was verified by molecular docking and molecular force. Furthermore, the chemical constituents of GHYSJ were analyzed by LC-MS and HPLC, the effects of GHYSJ on animal tissues were analyzed by H&E staining. An Aß-induced SH-SY5Y cellular model was established to validate the core pathways and targets predicted by network pharmacology and molecular docking. RESULTS: The results of the network pharmacology analysis revealed a total of 155 bioactive compounds capable of crossing the blood-brain barrier and interacting with 677 targets, among which 293 targets specifically associated with AD, which mainly participated in and regulated the amyloid aggregation pathway and PI3K/Akt signaling pathway, thereby treating AD. In addition, molecular docking analysis revealed a robust binding affinity between the principal bioactive constituents of GHYSJ and crucial targets implicated in AD. Our findings were further substantiated by in vitro experiments, which demonstrated that Liquiritigenin and Ginsenosides Rh4, crucial constituents of GHYSJ, as well as GHYSJ pharmaceutic serum, exhibited a significant down-regulation of BACE1 expression in Aß-induced damaged SH-SY5Y cells. This study provides valuable data and theoretical underpinning for the potential therapeutic application of GHYSJ in the treatment of AD and secondary development of GHYSJ prescription. CONCLUSION: Through network pharmacology, molecular docking, LC-MS, and cellular experiments, GHYSJ was initially confirmed to delay the progression of AD by regulating the expression of BACE1 in Amyloid aggregation pathway. Our observations provided valuable data and theoretical underpinning for the potential therapeutic application of GHYSJ in the treatment of AD.

Recent Advances of Seed-Mediated Growth of Metal Nanoparticles: from Growth to Applications.

Unprecedented advances in metal nanoparticle synthesis have paved the way for broad applications in sensing, imaging, catalysis, diagnosis, and therapy by tuning the optical properties, enhancing catalytic performance, and improving chemical and biological properties of metal nanoparticles. The central guiding concept for regulating the size and morphology of metal nanoparticles is identified as the precise manipulation of nucleation and subsequent growth, often known as seed-mediated growth methods. However, since the growth process is sensitive not only to the metal seeds but also to capping agents, metal precursors, growth solution, growth/incubation time, reductants, and other influencing factors, the precise control of metal nanoparticle morphology is multifactorial. Further, multiple reaction parameters are entangled with each other, so it is necessary to clarify the mechanism by which each factor precisely regulates the morphology of metal nanoparticles. In this review, to exploit the generality and extendibility of metal nanoparticle synthesis, the mechanisms of growth influencing factors in seed-mediated growth methods are systematically summarized. Second, a variety of critical properties and applications enabled by grown metal nanoparticles are focused upon. Finally, the current progress and offer insights on the challenges, opportunities, and future directions for the growth and applications of grown metal nanoparticles are reviewed.

An NIR fluorescent/photoacoustic dual-mode probe of NADPH for tumor imaging.

A novel probe was synthesized with a turn-on NIR fluorescent (NIRF)/photoacoustic (PA) response to NADPH, which was successfully applied in both monitoring intracellular NADPH and dual-modal imaging of tumor-bearing mice. It exhibits good potential in studying and understanding the tumor energy metabolism and treatment process related to NADPH.

Ultra-Small High-Entropy Alloy Nanoparticles: Efficient Nanozyme for Enhancing Tumor Photothermal Therapy.

High-entropy alloys nanoparticles (HEANPs) are receiving extensive attention due to their broad compositional tunability and unlimited potential in bioapplication. However, developing new methods to prepare ultra-small high-entropy alloy nanoparticles (US-HEANPs) faces severe challenges owing to their intrinsic thermodynamic instability. Furthermore, there are few reports on studying the effect of HEANPs in tumor therapy. Herein, the fabricated PtPdRuRhIr US-HEANPs act as bifunctional nanoplatforms for the highly efficient treatment of tumors. The US-HEANPs are engineered by the universal metal-ligand cross-linking strategy. This simple and scalable strategy is based on the aldol condensation of organometallics to form the target US-HEANPs. The synthesized US-HEANPs exhibit excellent peroxidase-like (POD-like) activity and can catalyze the endogenous hydrogen peroxide to produce highly toxic hydroxyl radicals. Furthermore, the US-HEANPs possess a high photothermal conversion effect for converting 808 nm near-infrared light into heat energy. In vivo and in vitro experiments demonstrated that under the synergistic effect of POD-like activity and photothermal action, the US-HEANPs can effectively ablate cancer cells and treat tumors. It is believed that this work not only provides a new perspective for the fabrication of HEANPs, but also opens the high-entropy nanozymes research direction and their biomedical application.

Biomolecule-mediated chiral nanostructures: a review of chiral mechanism and application.

The chirality of biomolecules is vital importance in biosensing and biomedicine. However, most biomolecules only have a chiral response in the ultraviolet region, and the corresponding chiral signal is weak. In recent years, inorganic nanomaterials can adjust chiral light signals to the visible and near-infrared regions and enhance optical signals due to their high polarizability and adjustable morphology-dependent optical properties. Nonetheless, inorganic nanomaterials usually lack specificity to identify targets, and have strong toxicity when applied in organisms. The combination of chiral biomolecules and inorganic nanomaterials offers a way to solve these problems. Because chiral biomolecules, such as DNA, amino acids, and peptides, have programmability, specific recognition, excellent biocompatibility, and strong binding force to inorganic nanomaterials. Biomolecule-mediated chiral nanostructures show specific recognition of targets, extremely low biological toxicity and adjustable optical activity by regulating, assembling and inducing inorganic nanomaterials. Therefore, biomolecule-mediated chiral nanostructures have received widespread attention, including chiral biosensing, enantiomers recognition and separation, biological diagnosis and treatment, chiral catalysis, and circular polarization of chiral metamaterials. This review mainly introduces the three chiral mechanisms of biomolecule-mediated chiral nanostructures, lists some important applications at present, and discusses the development prospects of biomolecule-mediated chiral nanostructures.

[Effects of betulinic acid at different concentrations on autophagy of human gastric cancer MGC-803 cells].

Objective: In this study, human gastric cancer MGC-803 cells were treated with betulinic acid at different concentrations to investigate its effect on cell autophagy. Methods: The human gastric cancer MGC-803 cells were divided into 4 groups, each group was set with 3 replicate. The control group was not treated with betulinic acid, the other three groups were added with final concentration of 10,20,30 mg/L betulinic acid, respectively. Cells were treated with betulinic acid for 48 h,qRT-PCR was applied to detect the effect of betulinic acid on mRNA expressions of autophagy-related genes in human gastric cancer MGC-803 cells. Western blot was performed to determine the protein expressions of cell autophagy-related genes after drug treatment. Immunofluorescence was used to detect the localization and expression of LC3 protein in MGC-803 cells after drug treatment. Results: Compared with the control group,in the concentration range of 10~30 mg/L, the mRNA expression of LC3 and Beclin-1 in human gastric cancer MGC-803 cells treated with betulinic acid were increased significantly, the expressions of Beclin-1 and LC3-Ⅱ protein were also increased significantly, while the expression of LC3-Ⅰ protein was decreased significantly. Among them, betulinic acid at the concentration of 30 mg/L showed the best effects. In addition, betulinic acid induced the LC3 protein in MGC-803 cells to form spot aggregates in the cytoplasm. Conclusion: At the concentrations of 10~30 mg/L, betulinic acid can induce autophagy in human gastric cancer MGC-803 cells.

[Effect of betulinic acid on proliferation of human gastric cancer MGC-803 cell line].

Objective: To investigate the effect of betulinic acid on the proliferation of human gastric cancer MGC-803 cells in vitro. Methods: Human gastric cancer MGC-803 cells were divided into 4 groups, each with 3 multiple holes. Control cells add betulinic acid at a concentration of 0 µg /ml, and the other three experimental groups were added with final concentration of 10, 20, 30 µg/ml Betulinic acid respectively. Cells in each group were incubated in a 5% CO2 incubator for 48 hours, and the Giemsa staining method and trypan blue exclusion method were used to detect the effect of betulinic acid on the cell clone formation rate and growth inhibition rate; EdU method and flow cytometry were used to detect cell proliferation and cell cycle changes; qRT-PCR and Western blot were used to detect the expressions of cell cycle regulators CCNB1 and CCND1. Results: Compared with the control group, the clone formation rate of human gastric cancer MGC-803 cells was significantly reduced (P＜0.01), the growth inhibition rate was significantly increased, and the cell proliferation ability was significantly decreased (P＜0.01); with the increase of betulinic acid concentration in each experimental group the proportion of cells in the G1 phase was gradually decreased, and the number of cells in S phase was increased significantly (P＜0.01); the mRNA and protein expression levels of cell cycle regulators CCNB1 and CCND1 were decreased significantly, and the 30 µg/ml betulinic acid treatment group performed best. Conclusion: At a final concentration of 10～30 µg/ml, betulinic acid can reduce the proliferation of human gastric cancer MGC-803 cells, inhibit cell growth, and down-regulate the expression of CCNB1 and CCND1 to block human gastric cancer MGC-803 cells in the S phase.

[Effects of betulinic acid on apoptosis of human gastric cancer MGC-803 cells].

Objective: The effects of betulinic acid (BA) on apoptosis of human gastric cancer MGC-803 cells was investigated by using human gastric cancer MGC-803 cells as experimental materials, and the basis for its clinical application was provided. Methods: The human gastric cancer MGC-803 cells were divided into 4 groups,each group was set with 3 replicates.The control group was MGC-803 cells without being added betulinic acid; the other 3 groups of experimental groups were treated with betulinic acid at final concentrations of 10, 20 and 30 µg /ml respectively. Cells were treated with betulinic acid of different concentrations for 48 h. Laser confocal microscope was used to observe morphological changes of MGC-803. The activities of Caspase-3 and Caspase-9 were detected by an assay kit. Flow cytometry was applied to determine mitochondrial membrane potential. The mRNA and protein levels of Caspase-3, Caspase-9 and Cyt c were also detected by qRT-PCR and Western blot, respectively. Results: Compared with the control group, the activities of Caspase-3 and caspase-9 were increased(P＜0.01), while the mitochondrial membrane potential was decreased significantly(P＜0.01). The mRNA and protein expressions of Caspase-3, caspase-9 and Cyt c were up-regulated significantly(P＜0.01). Conclusion: In the final concentration range of 10 ~ 30 µg/ml, betulinic acid can induce apoptosis of human gastric cancer MGC-803 cells by regulating the expression of Caspase-3, Caspase-9 and Cyt c.

[Effects of Betulinic acid on apoptosis in human gastric cancer SGC-7901 cells].

Objective: To investigate the effects of betulinic acid on apoptosis of human gastric cancer SGC-7901 cells. Methods: The human gastric cancer SGC-7901cells were divided in to 4 groups, and each group was set with 3 replicates. The SGC-7901cells in control group were not treated with betulinic acid; the other 3 experimental groups were treated with betulinic acid at the concentrations of 10, 20 and 30 mg/L, respectively; each group was incubated in a 5% carbon dioxide incubator for 48 h. Laser confocal microscope was used to observe morphological changes of SGC-7901 cells; Flow cytometry was applied to determine apoptosis rate and mitochondrial membrane potential. The mRNA and protein levels of Bcl-2, Bax and Caspase-3 were also detected by qRT-PCR and western blot respectively. Results: Compared with the control group, SGC-7901 cells in the treated group at final concentrations of 10, 20 and 30 mg/L shrinked, appeared apoptosis body along with nuclear splitting. The percentage of cells in early and advanced period of apoptosis were markedly increased (P＜0.05 or P＜0.01), mitochondrial membrane potential was obviously reduced (P＜0.05 or P＜0.01). qRT-PCR and western blot analysis showed that the mRNA and protein expressions of Bax and Caspase-3 were increased significantly (P＜0.01), while the expressions of Bcl-2 were decreased significantly (P＜0.01). Conclusion: Within a certain range of concentrations, betulinic acid induces cell apoptosis by regulating the expression of Bcl-2, Bax and Caspase-3 in human gastric cancer.