Erchen Decoction alleviates obesity-related hepatic steatosis via modulating gut microbiota-drived butyric acid contents and promoting fatty acid ß-oxidation.

ETHNOPHARMACOLOGICAL RELEVANCE: Erchen decoction (ECD) is a traditional Chinese medicine formula comprising six distinct herbs and has been documented to possess a protective effect against obesity. The study conducted previously demonstrated that ECD has the potential to effectively modulate the composition of gut microbiota and levels of short-chain fatty acids (SCFAs) in obese rat. However, the regulatory mechanism of ECD on gut microbiota and SCFAs and further improvement of obesity have not been thoroughly explained. AIM OF THE STUDY: The objective of this study was to examine the therapeutic effect and molecular mechanism of ECD in a rat model of high-fat diet (HFD) feeding. MATERIALS AND METHODS: Rats with HFD-induced obesity were treated with ECD. Upon completion of the study, serum and liver samples were procured to conduct biochemical, pathological, and Western blotting analyses. The investigation of alterations in the gut microbiota subsequent to ECD treatment was conducted through the utilization of 16S rRNA sequencing. The metabolic alterations in the cecal contents were examined through the utilization of mass spectrometry-ultraperformance liquid chromatography. RESULTS: ECD treatment improved lipid metabolic disorders and reduced hepatic steatosis in HFD-induced obese rats. Obese rat treated with ECD showed a higher abundance of SCFA-producing bacteria, including Lactobacillus, Bifidobacterium, and Butyricicoccus, and lower abundance of disease-related bacteria, such as Bacteroides, Parabacteroides, and Sediminibacterium. Additionally, ECD caused an increase in total SCFAs levels; in particular, butyric acid was dramatically increased in the HFD group. Rats treated with ECD also exhibited significantly increased butyric acid concentrations in the serum and liver. The subsequent reduction in histone deacetylase 1 expression and increase in acetyl-histone 3-lysine 9 (H3K9ac) levels contributed to the promotion of fatty acid ß-oxidation (FAO) in liver by ECD. CONCLUSION: This study demonstrates that ECD regulates the gut microbiota and promotes butyric acid production to ameliorate obesity-related hepatic steatosis. The mechanism might be related to the promotion of FAO via a butyric acid-mediated increase in H3K9ac levels in the liver.

ZiBuPiYin recipe ameliorates diabetes-associated cognitive decline by improving neuronal mitochondrial function in chronic psychologically stressed zucker diabetic fatty rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Zibu Piyin Recipe (ZBPYR) is a traditional Chinese medicine compound composed of 12 kinds of Chinese herbal medicines including red ginseng and yam. Long-term basic and clinical applications have proved that ZBPYR can prevent and treat cognitive dysfunction. Previous studies showed that chronic psychological stress can increase the risk of type 2 diabetes mellitus (T2DM), and lead to cognitive decline. Mitochondrial dysfunction plays a key role in chronic psychological stress-induced diabetes mellitus. While the mechanism of mitochondrial dysfunction and insulin resistance in diabetes-associated cognitive decline (DACD) is unclear. AIM OF THE STUDY: Our previous research found that a ZiBuPiYin recipe (ZBPYR) has significant pharmacological effects against DACD. The present study investigated changes in mitochondrial dysfunction in the brain and the mechanism of insulin resistance and mitochondrial damage to explore the relationship between neuronal mitochondrial dysfunction and insulin resistance in chronic psychologically stressed DACD rats. MATERIALS AND METHODS: Zucker diabetic fatty (ZDF) rats with spontaneous T2DM and rats with diabetic cognitive impairment that was induced by chronic psychological stress were used in in vivo experiments. PC12 cells that were damaged by rotenone were used for the in vitro experiment. RESULTS: The findings indicated that the number of mitochondria decreased, morphology and membrane potential were damaged, and reactive oxygen species increased in the cortex and hippocampus in psychologically stressed DACD rats. Protein kinase Cß2 (PKCß2) activation and insulin resistance were markedly induced by chronic psychological stress, together with decreases in peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) and mitochondrial fusion protein 2 (Mfn2). Furthermore, ZBPYR exerted protective effects both in in vivo and in vitro. CONCLUSION: Mitochondrial damage and insulin resistance were observed in the brain in chronic psychologically stressed DACD rats. The ZBPYR significantly improved brain mitochondrial damage and insulin resistance in chronic psychologically stressed DACD rats. These results provide novel insights for the development of ZBPYR as a traditional Chinese medicine for the treatment of chronic psychological stress and DACD.

Retraction Note to: Lupeol induces apoptosis and inhibits invasion in gallbladder carcinoma GBC-SD cells by suppression of EGFR/MMP-9 signaling pathway.

[This retracts the article DOI: 10.1007/s10616-014-9763-7.].

Liver mitochondria-associated endoplasmic reticulum membrane proteomics for studying the effects of ZiBuPiYin recipe on Zucker diabetic fatty rats after chronic psychological stress.

Type 2 diabetes mellitus (T2DM) is a complex metabolic disease with multiple etiologies, involving both genetic and environmental factors. With changes associated with modern life, increasing attention has been paid to chronic psychological stressors such as work stress. Chronic psychological stress can induce or aggravate diabetes mellitus, and conversely, with the deterioration of T2DM, patients often experience different degrees of depression, anxiety, and other negative emotions. In order to clarify the role of ZiBuPiYin recipe (ZBPYR) in regulating the liver mitochondria-associated endoplasmic reticulum membrane proteome to improve T2DM with chronic psychological stress, differentially expressed proteins (DEPs) were identified among Zucker lean littermates (control group), chronic psychological stress T2DM rats (model group), and ZBPYR administration rats (ZBPYR group) through iTRAQ with LC-MS/MS. Using Mfuzz soft clustering analysis, DEPs were divided into six different clusters. Clusters 1-6 contained 5, 68, 44, 57, 28, and 32 DEPs, respectively. Given that ZBPYR can alleviate T2DM symptoms and affect exploratory behavior during T2DM with chronic psychological stress, we focused on the clusters with opposite expression trends between model:control and ZBPYR:model groups. We screened out the DEPs in clusters 1, 3, and 4, which may be good candidates for the prevention and treatment of T2DM with chronic psychological stress, and further conducted bioinformatics analyses. DEPs were mainly involved in the insulin signaling pathway, oxidative phosphorylation, tricarboxylic acid cycle, amino acid metabolism, lysosome-related processes, and lipid metabolism. This may indicate the pathogenic basis of T2DM with chronic psychological stress and the potential therapeutic mechanism of ZBPYR. In addition, two key proteins, lysosome-associated protein (Lamp2) and tricarboxylic acid cycle-related protein (Suclg1), may represent novel biomarkers for T2DM with chronic psychological stress and drug targets of ZBPYR. Western blot analyses also showed similar expression patterns of these two proteins in liver MAMs of the model and ZBPYR groups.

Integrated Analyses of Microbiomics and Metabolomics Explore the Effect of Gut Microbiota Transplantation on Diabetes-Associated Cognitive Decline in Zucker Diabetic Fatty Rats.

Diabetes-associated cognitive decline (DACD), one of the complications of type 2 diabetes (T2DM), correlates significantly with the disorder in glycolipid metabolism, insulin/leptin resistance, and accumulation of ß-amyloid (Aß). Although gut microbiota transplantation (GMT), a novel non-invasive physiotherapy strategy, has been a promising intervention to alleviate the symptoms of T2DM, its protective effect on progressive cognitive decline remains elusive. Here, we transplanted the gut microbiota of healthy or cognitive decline donor rats into ZDF or LZ rats, and integrated microbiomics and metabolomics to evaluate the directional effect of the gut microbiota on the recipient rats. The basal metabolism phenotype changed in ZDF rats instead of in LZ rats. One possible mechanism is that the microbiota and metabolites alter the structure of the intestinal tract, stimulate the brain insulin and leptin signaling pathways, and regulate the deposition of Aß in the brain. It is worth noting that 10 species of genera, such as Parabacteroides, Blautia, and Lactobacillus, can regulate 20 kinds of metabolites, such as propanoic acid, acetic acid, and citramalic acid, and having a significant improvement on the cognitive behavior of ZDF rats. In addition, the correlation analysis indicated the gut microbiota and metabolites are highly associated with host phenotypes affected by GMT. In summary, our study indicates that altering the microbiota-gut-brain axis by reshaping the composition of gut microbiota is a viable strategy that has great potential for improving cognitive function and combatting DACD.

Conditional Knockout of Pdha1 in Mouse Hippocampus Impairs Cognitive Function: The Possible Involvement of Lactate.

Background and Purpose: Neurodegenerative diseases are associated with metabolic disturbances. Pyruvate dehydrogenase E1 component subunit alpha (PDHA1) is an essential component in the process of glucose metabolism, and its deficiency exists in various diseases such as Alzheimer's disease (AD), epilepsy, Leigh's syndrome, and diabetes-associated cognitive decline. However, the exact role of PDHA1 deficiency in neurodegenerative diseases remains to be elucidated. In this study, we explored the effect of PDHA1 deficiency on cognitive function and its molecular mechanism. Methods: A hippocampus-specific Pdha1 knockout (Pdha1 -/-) mouse model was established, and behavioral tests were used to evaluate the cognitive function of mice. Transmission electron microscopy (TEM) was performed to observe the morphological changes of the hippocampus. The lactate level in the hippocampus was measured. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting were used to explore the possible mechanism of the effect of PDHA1 on cognition. Results: Pdha1 knockout damaged the spatial memory of mice and led to the ultrastructural disorder of hippocampal neurons. Lactate accumulation and abnormal lactate transport occurred in Pdha1 -/- mice, and the cyclic AMP-protein kinase A-cAMP response element-binding protein (cAMP/PKA/CREB) pathway was inhibited. Conclusion: Lactate accumulation caused by PDHA1 deficiency in the hippocampus may impair cognitive function by inhibiting the cAMP/PKA/CREB pathway.

ZiBuPiYin Recipe Prevented and Treated Cognitive Decline in ZDF Rats With Diabetes-Associated Cognitive Decline via Microbiota-Gut-Brain Axis Dialogue.

Gut microbiota is becoming one of the key determinants in human health and disease. Shifts in gut microbiota composition affect cognitive function and provide new insights for the prevention and treatment of neurological diseases. Diabetes-associated cognitive decline (DACD) is one of the central nervous system complications of type 2 diabetes mellitus (T2DM). ZiBuPiYin recipe (ZBPYR), a traditional Chinese medicine (TCM) formula, has long been used for the treatment of T2DM and prevention of DACD. However, the contribution of ZBPYR treatment to the interaction between the gut microbiota and metabolism for preventing and treating DACD remains to be clarified. Here, we investigate whether the gut microbiota plays a key role in ZBPYR-mediated prevention of DACD and treatment of T2DM via incorporating microbiomics and metabolomics, and investigate the links between the microbiota-gut-brain axis interaction and the efficacy of ZBPYR in ZDF rats. In the current study, we found that ZBPYR treatment produced lasting changes in gut microbiota community and metabolites and remotely affected hippocampus metabolic changes, thereby improving memory deficits and reversing ß-amyloid deposition and insulin resistance in the brain of ZDF rats from T2DM to DACD. This may be related to a series of metabolic changes affected by gut microbiota, including alanine, aspartic acid, and glutamic acid metabolism; branched-chain amino acid metabolism; short-chain fatty acid metabolism; and linoleic acid/unsaturated fatty acid metabolism. In summary, this study demonstrates that prevention and treatment of DACD by ZBPYR partly depends on the gut microbiota, and the regulatory effects of bacteria-derived metabolites and microbiota-gut-brain axis are important protective mechanisms of ZBPYR.

Retraction: Oxymatrine Synergistically Potentiates the Antitumor Effects of Cisplatin in Human Gastric Cancer Cells.

[This retracts the article DOI: 10.7150/jca.28532.].

Fecal microbiota transplantation alters the susceptibility of obese rats to type 2 diabetes mellitus.

Obesity is one of the susceptibility factors for type 2 diabetes (T2DM), both of which could accelerate the aging of the body and bring many hazards. A causal relationship is present between intestinal microbiota and body metabolism, but how the microbiota play a role in the progression of obesity to T2DM has not been elucidated. In this study, we transplanted healthy or obese-T2DM intestinal microbiota to ZDF and LZ rats, and used 16S rRNA and targeted metabonomics to evaluate the directional effect of the microbiota on the susceptibility of obese rats to T2DM. The glycolipid metabolism phenotype could be changed bidirectionally in obese rats instead of in lean ones. One possible mechanism is that the microbiota and metabolites alter the structure of the intestinal tract, and improve insulin and leptin resistance through JAK2 / IRS / Akt pathway. It is worth noting that 7 genera, such as Lactobacillus, Clostridium and Roche, can regulate 15 metabolites, such as 3-indolpropionic acid, acetic acid and docosahexaenoic acid, and have a significant improvement on glycolipid metabolism phenotype. Attention to intestinal homeostasis may be the key to controlling obesity and preventing T2DM.

Effect of the ZiBuPiYin Recipe on Diabetes-Associated Cognitive Decline in Zucker Diabetic Fatty Rats After Chronic Psychological Stress.

BACKGROUND: Cognitive impairment is a complication of type 2 diabetes mellitus (T2DM) that affects the central nervous system (CNS). Studies have shown that chronic psychological stress may promote the development of T2DM into diabetes-associated cognitive decline (DACD). Previously, cognitive impairment in T2DM was correlated predominantly with insulin resistance in the medial prefrontal cortex (mPFC). AIMS: We examined the effect of the ZiBuPiYin recipe (ZBPYR) on Zucker diabetic fatty (ZDF) rats and explored the impact of chronic stress on altered ß-amyloid (Aß) metabolism through insulin receptor substrate (IRS) 1/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway after the induction of chronic psychological stress. MAIN METHODS: After chronic psychological stress and drug treatment, cognitive function was observed via behavioral experiments. The activation of the hypothalamus-pituitary-adrenal (HPA) axis and levels of Aß were detected by enzyme-linked immunosorbent assay, and the expression of related proteins was evaluated by Western blotting. KEY FINDINGS: ZBPYR treatment significantly decreased anxious-like behaviors and plasma corticosterone (CORT) levels, and ameliorated learning and memory impairments of ZDF rats after chronic psychological stress. ZBPYR also reduced the deposition of Aß in the mPFC, improved brain insulin resistance, and modulated the mTOR-autophagy pathway. SIGNIFICANCE: ZBPYR may be a potential therapeutic application for the treatment of DACD induced by chronic psychological stress.

S-adenosylmethionine induces apoptosis and cycle arrest of gallbladder carcinoma cells by suppression of JAK2/STAT3 pathways.

S-adenosylmethionine (SAM) is a naturally occurring physiologic molecule found ubiquitously in all mammalian cells and an essential compound in many metabolic pathways. It has been reported to possess many pharmacological properties including cancer-preventive and anticancer effects. However, the precise molecular mechanism involved in its anticancer effect is not yet clear. The present study is conducted to investigate the anticancer activity and the underlying mechanisms of SAM on human gallbladder cancer cells (GBC-SD and SGC-996) in vitro and in vivo. Cells were dealt with SAM and subjected to cell viability, colony formation, Hoechst staining, apoptosis, cycle arrest, western blot, and xenograft tumorigenicity assay. Experimental results showed that SAM could significantly inhibit the growth and proliferation and induce the apoptosis as well as cell cycle arrest in G0/G1 phase of GBC-SD and SGC-996 cells in a dose-dependent manner in vitro. The expression levels of p-JAK2, p-STAT3, Mcl-1, and Bcl-XL were significantly downregulated. In addition, inhibition of the JAK2/STAT3 pathway significantly enhanced the anti-apoptotic effect of SAM, suggesting the key roles of JAK2/STAT3 in the process. More importantly, our in vivo studies demonstrated that administration of SAM could significantly decrease the tumor weight and volume and immunohistochemistry analysis proved the downregulation of p-JAK2 and p-STAT3 in tumor tissues following SAM treatment, consistent with our in vitro results. In summary, our findings indicated that SAM can inhibit cell proliferation and induce apoptosis as well as cycle arrest of GBC cells by suppression of JAK2/STAT3 pathways and the dramatic effects of SAM hinting that SAM might be a useful therapeutic option for patients suffering from gallbladder cancer.

S-Adenosylmethionine synergistically enhances the antitumor effect of gemcitabine against pancreatic cancer through JAK2/STAT3 pathway.

Gemcitabine (GEM) has been widely used for pancreatic cancer (PC) treatment but limited by the development of drug resistance. The agents that reverse its resistance and improve the chemo-sensitivity are urgently needed. S-Adenosylmethionine (SAM) is a precursor for polyamine biosynthesis in mammalian cells and plays a key role in biological transmethylation events. It is reported that SAM could be used as a therapeutic reagent for cancer treatments. In this study, we investigated the chemo-sensitization of SAM to potentiate the antitumor effect of GEM in PC. After treating PC cells with GEM and/or SAM, different subsequent experiments were performed. Results showed that SAM plus GEM could significantly inhibit the growth and proliferation of PC cells, and SAM acts synergistically with GEM. The combinative treatment could induce cell apoptosis and inhibit invasion and migration through JAK2/STAT3 inactivation. Inhibition of JAK2/STAT3 pathway significantly enhanced the pro-apoptotic effect of SAM, suggesting the key roles of JAK2/STAT3 in the process. Moreover, co-treatment with GEM and SAM exhibited more efficient inhibition of tumor weight and volume on PANC-1 xenograft mouse model compared to GEM or SAM alone and has no significant effect on the function of the liver and kidney. In general, this study indicated that SAM synergistically enhanced the antitumor effect of GEM against PC through suppressed JAK2/STAT3 pathway, and SAM is applicable as a promising agent to improve the sensitivity of PC to GEM.

Oxymatrine Synergistically Potentiates the Antitumor Effects of Cisplatin in Human Gastric Cancer Cells.

Cisplatin (CDDP) has been extensively used for gastric cancer (GC) treatment but limited by drug resistance and severe toxicity. The chemo-sensitizers that enhance its efficiency and overcome its limitation are urgently needed. Oxymatrine (OMT), a primary active ingredient from the dry roots of Sophora favescens, has shown powerful anti-cancer property with little side-effect. In this study, we explored the chemo-sensitization of OMT to potentiate the anti-tumor effect of CDDP. GC cell lines were dealt with OMT and/or CDDP and then subjected to different experimental methods. We found that OMT could significantly potentiate the CDDP-caused BGC-823 and SGC7901 cells viability loss, and OMT acts synergistically with CDDP. The combinative treatment could arrest cell cycle in G0/G1 phase by increasing p21, p27 and decreasing cyclin D1, and induced apoptosis by ROS generation and AKT/ERK inactivation. Inhibition of ROS respectively reversed the cell death induced by OMT and/or CDDP, suggesting the pivotal roles of ROS in the process. Moreover, OMT enhanced the antitumor effects of CDDP in nude mice bearing BGC823 tumor xenografts in vivo. Taken together, this study highlights that the co-treatment with OMT and CDDP exerted synergistic antitumor effects in GC cells, and that these effects may be mediated by ROS generation and inactivation of the AKT/ERK pathways.

Spatial receptive field shift by preceding cross-modal stimulation in the cat superior colliculus.

KEY POINTS: It has been known for some time that sensory information of one type can bias the spatial perception of another modality. However, there is a lack of evidence of this occurring in individual neurons. In the present study, we found that the spatial receptive field of superior colliculus multisensory neurons could be dynamically shifted by a preceding stimulus in a different modality. The extent to which the receptive field shifted was dependent on both temporal and spatial gaps between the preceding and following stimuli, as well as the salience of the preceding stimulus. This result provides a neural mechanism that could underlie the process of cross-modal spatial calibration. ABSTRACT: Psychophysical studies have shown that the different senses can be spatially entrained by each other. This can be observed in certain phenomena, such as ventriloquism, in which a visual stimulus can attract the perceived location of a spatially discordant sound. However, the neural mechanism underlying this cross-modal spatial recalibration has remained unclear, as has whether it takes place dynamically. We explored these issues in multisensory neurons of the cat superior colliculus (SC), a midbrain structure that involves both cross-modal and sensorimotor integration. Sequential cross-modal stimulation showed that the preceding stimulus can shift the receptive field (RF) of the lagging response. This cross-modal spatial calibration took place in both auditory and visual RFs, although auditory RFs shifted slightly more. By contrast, if a preceding stimulus was from the same modality, it failed to induce a similarly substantial RF shift. The extent of the RF shift was dependent on both temporal and spatial gaps between the preceding and following stimuli, as well as the salience of the preceding stimulus. A narrow time gap and high stimulus salience were able to induce larger RF shifts. In addition, when both visual and auditory stimuli were presented simultaneously, a substantial RF shift toward the location-fixed stimulus was also induced. These results, taken together, reveal an online cross-modal process and reflect the details of the organization of SC inter-sensory spatial calibration.

Neuronal delivery of Hedgehog directs spatial patterning of taste organ regeneration.

How organs maintain and restore functional integrity during ordinary tissue turnover or following injury represents a central biological problem. The maintenance of taste sensory organs in the tongue was shown 140 years ago to depend on innervation from distant ganglion neurons, but the underlying mechanism has remained unknown. Here, we show that Sonic hedgehog (Shh), which encodes a secreted protein signal, is expressed in these sensory neurons, and that experimental ablation of neuronal Shh expression causes loss of taste receptor cells (TRCs). TRCs are also lost upon pharmacologic blockade of Hedgehog pathway response, accounting for the loss of taste sensation experienced by cancer patients undergoing Hedgehog inhibitor treatment. We find that TRC regeneration following such pharmacologic ablation requires neuronal expression of Shh and can be substantially enhanced by pharmacologic activation of Hedgehog response. Such pharmacologic enhancement of Hedgehog response, however, results in additional TRC formation at many ectopic sites, unlike the site-restricted regeneration specified by the projection pattern of Shh-expressing neurons. Stable regeneration of TRCs thus requires neuronal Shh, illustrating the principle that neuronal delivery of cues such as the Shh signal can pattern distant cellular responses to assure functional integrity during tissue maintenance and regeneration.

Metformin synergistically enhances antitumor activity of cisplatin in gallbladder cancer via the PI3K/AKT/ERK pathway.

Metformin (Met) is a widely used antidiabetic drug and has demonstrated interesting anticancer effects in various cancer models, alone or in combination with chemotherapeutic drugs. The aim of the present study is to investigate the synergistic effect of Met with cisplatin (Cis) on the tumor growth inhibition of gallbladder cancer cells (GBC-SD and SGC-996) and explore the underlying mechanism. Cells were treated with Met and/or Cis and subjected to cell viability, colony formation, apoptosis, cell cycle, western blotting, xenograft tumorigenicity assay and immunohistochemistry. The results demonstrated that Met and Cis inhibited the proliferation of gallbladder cancer cells, and combination treatment with Met and Cis resulted in a combination index < 1, indicating a synergistic effect. Co-treatment with Met and Cis caused G0/G1 phase arrest by upregulating P21, P27 and downregulating CyclinD1, and induced apoptosis through decreasing the expression of p-PI3K, p-AKT, and p-ERK. In addition, pretreatment with a specific AKT activator (IGF-1) significantly neutralized the pro-apoptotic activity of Met + Cis, suggesting the key role of AKT in this process. More importantly, in nude mice model, Met and Cis in combination displayed more efficient inhibition of tumor weight and volume in the SGC-996 xenograft mouse model than Met or Cis alone. Immunohistochemistry analysis suggests the combinations greatly suppressed tumor proliferation, which is consistent with our in vitro results. In conclusion, our findings indicate that the combination therapy with Met and Cis exerted synergistic antitumor effects in gallbladder cancer cells through PI3K/AKT/ERK pathway, and combination treatment with Met and Cis would be a promising therapeutic strategy for gallbladder cancer patients.

PM2.5 obtained from urban areas in Beijing induces apoptosis by activating nuclear factor-kappa B.

BACKGROUND: Particulate matter (PM), which has adverse effects on citizen health, is a major air pollutant in Beijing city. PM2.5 is an indicator of PM in urban areas and can cause serious damage to human health. Many epidemiological studies have shown that nuclear factor-kappa B (NF-κB) is involved in PM2.5-induced cell injury, but the exact mechanisms are not well understood. METHODS: The cytotoxic effects of PM2.5 at 25-1600 µg/ml for 24 h were determined by MTT assay in Chinese hamster ovary cells (CHO) cells. Flow cytometry was used to determine the apoptosis rate induced by PM2.5. The destabilized enhanced green fluorescent protein (d2EGFP) green fluorescent protein reporter system was used to determine the NF-κB activity induced by PM2.5. The expression of pro-apoptotic Bcl-2-associated death promoter (BAD) proteins induced by PM2.5 was determined by western blotting to explore the relationship between PM2.5 and the NF-κB signaling pathway and to determine the toxicological mechanisms of PM2.5. RESULTS: PM2.5 collected in Beijing urban districts induces cytotoxic effects in CHO cells according to MTT assay with 72.28% cell viability rates even at 200 µg/ml PM2.5 and flow cytometry assays with 26.97% apoptosis rates at 200 µg/ml PM2.5. PM2.5 increases the activation levels of NF-κB, which have maintained for 24 h. 200 µg/ml PM2.5 cause activation of NF-κB after exposure for 4 h, the activation peak appears after 13.5 h with a peak value of 25.41%. The average percentage of NF-κB activation in whole 24 h is up to 12.9% by 200 µg/ml PM2.5. In addition, PM2.5 decreases the expression level of the pro-apoptotic protein BAD in a concentration-dependent manner. CONCLUSIONS: PM2.5 induces NF-κB activation, which persists for 24 h. The expression of pro-apoptotic protein BAD decreased with increased concentrations of PM2.5. These findings suggest that PM2.5 plays a major role in apoptosis by activating the NF-κB signaling pathway and reducing BAD protein expression.