Lipid nanoparticle encapsulated oleic acid induced lipotoxicity to hepatocytes via ROS overload and the DDIT3/BCL2/BAX/Caspases signaling in vitro and in vivo.

BACKGROUND: To date, Non-alcoholic fatty liver disease (NAFLD) is one of the most common liver disease associated with clinical complications. Dietary fatty acids have been suggested to be involved in preventing or reversing the accumulation of hepatic fat. However, contradicting roles of monounsaturated fatty acids to the liver have been implicated in various human and murine models, mainly due to the insolubility nature of fatty acids. METHODS: High pressure homogenization methods were used to fabricate oleic acid embedded lipid nanoparticles (OALNs). The in vitro and in vivo models were used to validate the physiological effect of this OALNs via various cellular and molecular approaches including cell viability essay, fluorescent staining, electron microscope, RNAseq, qPCR, Western blots, and IHC staining. RESULTS: We successfully fabricated OALNs with enhanced stability and solubility. More importantly, lipid accumulation was successfully induced in hepatocytes via the application of OALNs in a dose-dependent manner. Overload of OALNs resulted in ROS accumulation and apoptosis of hepatocytes dose-dependently. With the help of transcriptome sequencing and traditional experimental approaches, we demonstrated that the lipotoxic effect induced by OALNs was exerted via the DDIT3/BCL2/BAX/Caspases signaling. Moreover, we also verified that OALNs induced steatosis and subsequent apoptosis in the liver of mice via the activation of DDIT3 in vivo. CONCLUSIONS: In all, our results established a potential pathogenic model of NAFLD for further studies and indicated the possible involvement of DDIT3 signaling in abnormal steatosis process of the liver.

CFTR is a negative regulator of γδ T cell IFN-γ production and antitumor immunity.

CFTR, a chloride channel and ion channel regulator studied mostly in epithelial cells, has been reported to participate in immune regulation and likely affect the risk of cancer development. However, little is known about the effects of CFTR on the differentiation and function of γδ T cells. In this study, we observed that CFTR was functionally expressed on the cell surface of γδ T cells. Genetic deletion and pharmacological inhibition of CFTR both increased IFN-γ release by peripheral γδ T cells and potentiated the cytolytic activity of these cells against tumor cells both in vitro and in vivo. Interestingly, the molecular mechanisms underlying the regulation of γδ T cell IFN-γ production by CFTR were either TCR dependent or related to Ca2+ influx. CFTR was recruited to TCR immunological synapses and attenuated Lck-P38 MAPK-c-Jun signaling. In addition, CFTR was found to modulate TCR-induced Ca2+ influx and membrane potential (Vm)-induced Ca2+ influx and subsequently regulate the calcineurin-NFATc1 signaling pathway in γδ T cells. Thus, CFTR serves as a negative regulator of IFN-γ production in γδ T cells and the function of these cells in antitumor immunity. Our investigation suggests that modification of the CFTR activity of γδ T cells may be a potential immunotherapeutic strategy for cancer.

Chrysanthemum ethanol extract induced loss of Kupffer cells via the mitochondria-dependent apoptotic pathway.

Chrysanthemum has been viewed as an important traditional Chinese medicine (TCM) with a long history. Research studies indicated many potential pharmaceutical effects of chrysanthemum extract. However, hardly any investigation has been performed to describe its toxicity. In this study, acute application of chrysanthemum ethanol extract (CEE, 300 mg kg-1) was found to induce apoptosis of hepatic Kupffer cells in vivo. CEE was also observed to induce apoptosis of RAW264.7 cells in a dose- and time-dependent manner. Further analysis using flow cytometry and western blotting revealed that CEE induced apoptosis of RAW264.7 cells via a mitochondria-dependent pathway. After a HPLC combined screening assay, we narrowed down the toxicity caused by the petroleum extract of CEE (CEE-PE, 66 µg mL-1). In vivo effects of CEE-PE were also tested in mice. Additionally, nine potential toxic compounds were isolated and identified from CEE-PE. In all, we found that components with small polarities in CEE could induce apoptosis of Kupffer cells and macrophages via a mitochondrial dependent pathway, which might draw attention to the safety issues of everyday use of chrysanthemum.

Nanoparticle-Encapsulated Liushenwan Could Treat Nanodiethylnitrosamine-Induced Liver Cancer in Mice by Interfering With Multiple Critical Factors for the Tumor Microenvironment.

We previously isolated an ethanol fraction of LSW (Liushenwan pill, a traditional Chinese medicine) which has been shown to prevent and treat liver cancer induced by nanodiethylnitrosamine (nanoDEN) in mice. In the present study, we utilized a high-pressure microfluidics technique to generate LSW lipid nanoparticles (nano-LSW) to reduce its toxicity, and enhance its inhibitory effect on tumor growth, and further evaluate its therapeutic effect using a nanoDEN-induced mouse model of liver cancer. Our in vitro results indicated that nano-LSW-low could induce apoptosis in HepG2 cells, but exhibited low toxicity in L02 cells. Furthermore, the in vivo results indicated that nano-LSW-low exerted minimal or no damage to normal hepatocytes, kidney, and small intestine tissues. In addition, our results showed that at the 20th week, the inflammatory infiltration in the mice in the model group increased severely, and partial pimelosis and fibrosis occurred. In contrast, the liver tissues in the mice treated with nano-LSW exhibited only slight inflammatory infiltration, without pimelosis and fibrosis. At the 30th week, 4 out of 5 liver tissues in the model group showed hyperplastic nodules by hematoxylin and eosin (H&E) staining. However, the liver tissues in the nano-LSW treatment group did not showed hyperplastic nodules. Immunohistochemical staining showed that, in contrast to the model group, the levels of COX-2, PCNA, ß-catenin, and HMGB1 protein expressions were significantly lower in the nano-LSW-low group at the 20th and 30th week. Compared to model group, the COX-2, TNF-α, Smad-2, and TGF-ß1 mRNA levels obviously decreased in the liver tissue after the nano-LSW-low treatment. Taken together, nano-LSW-low may serve as a potent therapeutic agent for preventing liver cancer by interfering with multiple critical factors for the tumor microenvironment during oncogenesis.

Dehydrocostus lactone inhibits cell proliferation and induces apoptosis by PI3K/Akt/Bad and ERS signalling pathway in human laryngeal carcinoma.

The anti-cancer effect of dehydrocostus lactone (DHL) derived from Saussurea costus (Falc.) Lipech against laryngeal carcinoma was assessed. The cytotoxic activity of DHL against laryngeal carcinoma is still obscure. Therefore, our study investigated the role of DHL in the growth inhibition of laryngeal carcinoma in vitro and in vivo, and the molecular mechanism of DHL-induced apoptosis in cancer cells of the larynx. The results showed that DHL inhibits the viability, migration and proliferation of Hep-2 and TU212 cells with little toxic effects on human normal larynx epithelial HBE cell line. Flow cytometry analysis (FAC) analysis and staining assay (Hoechst 33258) indicated that DHL stimulated Hep-2 and TU212 cell apoptosis in a dose-dependent manner. Mechanistically, DHL is capable of inhibiting Hep-2 and TU212 cell viability via promoting p53 and P21 function, meanwhile DHL dose-dependently induces Hep-2 and TU212 cells apoptosis via activating mitochondrial apoptosis by inhibiting PI3K/Akt/Bad pathway and stimulating endoplasmic reticulum stress-mediated apoptosis pathway. In vivo, DHL inhibited the growth of the Hep-2 nude mouse xenograft model and observed no significant signs of toxicity in the organs of nude mice. In vivo experiments further confirmed the anti-cancer effect of DHL on laryngeal carcinoma cells in vitro, and DHL-treated nude mice can reduce the volume of tumours. Together, our study indicated that DHL has the potential to inhibit human laryngeal carcinoma via activating mitochondrial apoptosis pathway by inhibiting PI3K/Akt/Bad signalling pathway and stimulating endoplasmic reticulum stress-mediated apoptosis pathway, providing a strategy for the treatment of human laryngeal carcinoma.

Effect of electroacupuncture on relieving central post-stroke pain by inhibiting autophagy in the hippocampus.

INTRODUCTION: To explore the underlying mechanism of electroacupuncture (EA) treatment on central post-stroke pain (CPSP), and provide basic evidence for the EA treatment on CPSP. METHODS: Firstly, 40 male SD rats were successfully established with a model of CPSP, under the intervention of different EA frequencies (2 Hz and 15 Hz) and fluoxetine (5 ml/kg and 0.4 mg/ml), whose brain tissue was then removed for paraffin-embedded sectioning; secondly, LPS induced the primary brain cells in the hippocampus to cause inflammation model which were added NS398 (inhibitor of COX-2) and DKK-1 (inhibitor of ß-catenin) later. The lesion sites of brain tissue were observed by Nissl staining and Transmission Electron Microscope (TEM) and autophagy-related proteins (LC3B, p62, LAMP-1), COX-2 and ß-catenin were detected by Western Blot and immunohistochemical staining. Finally, the correlation between LC3B, COX-2, and ß-catenin was calculated by multispectral quantification. RESULTS: (1) In the EA group (15 Hz), the number of Nissl bodies increased, autophagy-related protein LC3B-Ⅱ/Ⅰ, LAMP-1, COX-2, and ß-catenin was lowly expressed, p62 was highly expressed; (2) COX-2, ß-catenin and LC3B are positively correlated with each other (COX-2 & ß-catenin: r = 0.923; COX-2 & LC3B: r = 0.818; ß-catenin & LC3B: r = 0.801); (3) Nissl bodies of primary brain cells of the hippocampus under LPS were like animal experiments; after addition of DKK-1, high expression of ß-catenin and COX-2 induced by LPS was significantly down-regulated, and LC3B-II/I was significantly down-regulated, and p62 protein only had up-regulation trend; after addition of NS398, COX-2 and LC3B-II/I was significantly down-regulated. CONCLUSION: EA may inhibit autophagy in the hippocampus by reducing ß-catenin/COX-2 protein expression and effectively alleviating CPSP. SIGNIFICANCE STATEMENT: Previous studies have found that EA can reduce the expression of NK-1R in damaged rats by inhibition of COX-2 and ß-catenin loops, which controls the activation of glial cells in the damaged area and the apoptosis of neuronal cells, and alleviated pain. In the male SD rat model, we evaluated this effect that EA inhibits autophagy in the hippocampus by reducing ß-catenin/COX-2 protein expression in the brain tissue. In addition, we assessed expression levels of autophagy-related proteins and genes on the inflammatory primary brain cells model. From the experiment, we found EA may inhibit autophagy in the hippocampus by reducing ß-catenin/COX-2 protein expression. These findings provide a foundation for the interpretation of the mechanism of EA on relieving CPSP in clinical practice.

AMPK-ERK/CARM1 Signaling Pathways Affect Autophagy of Hepatic Cells in Samples of Liver Cancer Patients.

Liver cancer is one of the most common malignant tumors, with the death rate ranking fourth among all types of cancer. Over the past few decades, several studies have reported that liver tumorigenesis is associated with dysfunction in autophagy. However, the detailed mechanism remains unclear. In this paper, we used tissue micro-array (TMA) of liver cancer to detect proteins associated with the regulation of autophagic signaling in non-cancerous and cancerous regions by immunohistochemical staining. Those proteins contained 4-HNE, p-AMPK, Erk1/2, p-Erk1/2, CARM1, TFEB, LAMP1, and p62. According to the degrees of tumor differentiation in patients (well differentiated group vs. moderately and poorly differentiated group), we analyzed each protein's expression in the ratio of the "cancerous region/non-cancerous region" in two groups. Current data showed that there were AMPK-ERK/CARM1 autophagic signaling pathways during the formation of liver cancer. The above-mentioned changes in signals indicated an upregulation of autophagy in cancerous regions, which means overactivated autophagy plays an important role in liver cancer.

Solid lipid nanoparticle induced apoptosis of macrophages via a mitochondrial-dependent pathway in vitro and in vivo.

Background and aims: Lipid nanoparticles (LNs) are widely applied in drug delivery systems because they can incorporate and stabilize lipophilic and hydrophilic molecules. LNs are generally considered quite safe and convenient for in vivo applications. However, we previously observed that certain types of LNs could cause a loss of Kupffer cells, a kind of resident macrophage in the liver. As a result, we investigated the details of this phenomenon. Methods: MTT assay, Annexin-V-FITC/PI double staining, JC-1 staining, flow cytometry, Western blot and transmission electron microscopy were used in cell-based experiments. Additionally, serum biochemical analyses, H&E staining and immunofluorescence staining were performed to detect the acute and chronic changes of tissue structure and the number of Kupffer cells in mouse liver tissue samples. Results: Application of LN depolarized and swelled the mitochondria of Raw264.7 cells, and disrupted the balance of Bax/Bcl-2. This led to cleavage and activation of caspase-3 and PARP, and then induced apoptosis of Raw264.7 cells. In addition, either acute or chronic applications of LN were sufficient to disrupt the structure of the hepatic portal vein and reduce the number of Kupffer cells in mice. Conclusions: LNs could induce apoptosis of macrophages through a mitochondrial-dependent pathway.

The Ethanol Supernatant Extracts of Liushenwan Could Alleviate Nanodiethylnitrosamine-Induced Liver Cancer in Mice.

Liver cancer is one of the leading causes of cancerous deaths worldwide. At present, the treatment of hepatocellular carcinoma (HCC) remains to be a problem globally. Liushenwan (LSW), an ancient Chinese medicine previously used to treat localized infections, was recently reported to possess anticancer activity. Here in this study, we aim to examine the effect of LSW-ET (LSW-ET is the supernatant fraction of LSW from ultrasound assisted ethanol extraction) in prevention and treatment on nanodiethylnitrosamine- (nanoDEN-) induced HCC in mice. In nanoDEN-induced HCC mice treated with LSW-ET by oral (po) or intragastric gavage (ig), we observed an alleviation of serum ALT and AST levels, amelioration in histopathological stainings, and an inhibition in liver tumor growth. In addition, compared with the nanoDEN group, downregulation of multiple pivotal factors (COX-2, ß-catenin, PCNA, and HMGB-1) was observed in LSW-ET-po and LSW-ET-ig groups. Taken together, the delivery of LSW-ET by oral could be a potential prevention and treatment of liver cancer.

The dark side of "the force" - lipid nanoparticles enhance the oncogenesis of diethylnitrosamine and result in liver cancer in mice.

Nano-carriers, especially lipid nanoparticles have been used widely in "a good manner", for instance in the treatment of cancer, by enhancing the targetability and reducing required dose. Here in the contrary, we presented a new possibility: nanoDEN, a nanoparticle-packed "bad guy", which is more effective and efficient in generating liver tumor in mice. We have shown that nanoDEN, same as diethylnitrosamine (DEN), induced overexpression of multiple pivotal factors (including COX-2, ß-catenin and PCNA) during oncogenesis. Moreover, nanoDEN increased the apoptosis of liver cells compared with DEN alone. This apoptotic effect of nanoDEN is more efficient on normal cells than on cancer cells. Taken into consideration the fact that there are endogenous nanoparticles naturally formed inside our body, our research enlarged our views of all the aspects of oncogenic chemicals, while also established a better method of producing animal model of liver cancer, which has future investigational and therapeutical potential.

Chronic high-dosage fish oil exacerbates gut-liver axis injury in alcoholic steatohepatitis in mice: the roles of endotoxin and IL-4 in Kupffer cell polarization imbalance.

In the present study, intestinal tight junctions (TJs) and Kupffer cell polarization were investigated in an alcoholic steatohepatitis (ASH) mouse model to uncover the potential side effects of overexposure to fish oil or omega-3 fatty acids. The mice were fed ad libitum with a liquid diet containing ethanol and fish oil. In the meantime, ethanol was given every 5-7 days by gavage to simulate binge drinking. After the 7th binge, steatosis, necrosis, inflammatory infiltration, and bridging fibrosis were observed in the liver by histological staining. After the 13th binge, the inducers, markers and other downstream genes/proteins of the Kupffer cell M1/M2 phenotype in the liver, serum, and small intestine were analysed. The results suggested that a chronic high dosage of fish oil alone reduced the mRNA levels of most genes tested and showed a tendency to damage the intestinal zonula occludens-1 localization and reduce the number of M2 Kupffer cells. Meanwhile, the combination of fish oil and ethanol damaged the intestinal TJs, resulting in an increased endotoxin level in the liver. Gut-derived endotoxin polarized Kupffer cells to the M1 phenotype, whereas the number of cells with the M2 phenotype (markers: CD163 and CD206) was decreased. Interleukin-4 (IL-4), an M2 Kupffer cell inducer, was also decreased. Moreover, in vitro experiments showed that IL-4 reversed eicosapentaenoic acid-induced CD163 and CD206 mRNA suppression in RAW 264.7 cells. Overall, our results showed that a chronic high dosage of fish oil exacerbated gut-liver axis injury in alcoholic liver disease in mice, and endotoxin/IL-4-induced Kupffer cell polarization imbalance might play an important role in that process.

Nutmeg oil alleviates chronic inflammatory pain through inhibition of COX-2 expression and substance P release in vivo.

BACKGROUND: Chronic pain, or sometimes referred to as persistent pain, reduces the life quality of patients who are suffering from chronic diseases such as inflammatory diseases, cancer and diabetes. Hence, herbal medicines draw many attentions and have been shown effective in the treatment or relief of pain. METHODS AND RESULTS: Here in this study, we used the CFA-injected rats as a sustainable pain model to test the anti-inflammatory and analgesic effect of nutmeg oil, a spice flavor additive to beverages and baked goods produced from the seed of Myristica fragrans tree. CONCLUSIONS: We have demonstrated that nutmeg oil could potentially alleviate the CFA-injection induced joint swelling, mechanical allodynia and heat hyperanalgesia of rats through inhibition of COX-2 expression and blood substance P level, which made it possible for nutmeg oil to be a potential chronic pain reliever.

Mouse hepatic neoplasm formation induced by trace level and low frequency exposure to diethylnitrosamine through ß-catenin signaling pathway.

It has been reported that massive levels or/and high frequency exposure of diethylnitrosamine could induce hepatic neoplasm. However, it would be more interesting to figure out the hepatotoxic effects of diethylnitrosamine exposure at trace level and low frequency, which could be more common in our daily life. We found that both the mRNA and protein expression levels of ß-catenin were aberrant in all liver tissues, accompanied by inflammation, steatosis, fibrosis and hepatic neoplasm after 10-week exposure of diethylnitrosamine (dissolved in sesame oil, 0.16 mmol per kg body weight) to mice. In addition, gradual increase in the mRNA expression of several pivotal risk factors (TNF-α, COX-2, PPAR-γ, AP-2, Smad-2, TGF-ß1, and C-myc), as well as their protein expression levels, were associated with the aberrant expression or/and nucleus localization of ß-catenin. Altogether, our results show that long-term diethylnitrosamine exposure at trace amounts and low frequency can also induce hepatotoxicity (including inflammation, steatosis and fibrosis) and consequently aberrant activation of ß-catenin which in turn plays an important role in the initiation and promotion of liver tumors.

Mechanosensitivity of wild-type and G551D cystic fibrosis transmembrane conductance regulator (CFTR) controls regulatory volume decrease in simple epithelia.

Mutations of cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial ligand-gated anion channel, are associated with the lethal genetic disease cystic fibrosis. The CFTR G551D mutation impairs ATP hydrolysis and thereby makes CFTR refractory to cAMP stimulation. Both wild-type (WT) and G551D CFTR have been implicated in regulatory volume decrease (RVD), but the underlying mechanism remains incompletely understood. Here, we show that the channel activity of both WT and G551D CFTR is directly stimulated by mechanical perturbation induced by cell swelling at the single-channel, cellular, and tissue levels. Hypotonicity activated CFTR single channels in cell-attached membrane patches and WT-CFTR-mediated short-circuit current (Isc) in Calu-3 cells, and this was independent of Ca(2+)and cAMP/PKA signaling. Genetic suppression and ablation but not G551D mutation of CFTR suppressed the hypotonicity- and stretch-inducedIscin Calu-3 cells and mouse duodena. Moreover, ablation but not G551D mutation of the CFTR gene inhibited the RVD of crypts isolated from mouse intestine; more importantly, CFTR-specific blockers markedly suppressed RVD in both WT- and G551D CFTR mice, demonstrating for the first time that the channel activity of both WT and G551D CFTR is required for epithelial RVD. Our findings uncover a previously unrecognized mechanism underlying CFTR involvement in epithelial RVD and suggest that the mechanosensitivity of G551D CFTR might underlie the mild phenotypes resulting from this mutation.-Xie, C., Cao, X., Chen, X, Wang, D., Zhang, W. K., Sun, Y., Hu, W., Zhou, Z., Wang, Y., Huang, P. Mechanosensitivity of wild-type and G551D cystic fibrosis transmembrane conductance regulator (CFTR) controls regulatory volume decrease in simple epithelia.

Cytotoxicity of polyaniline nanomaterial on rat celiac macrophages in vitro.

Polyaniline nanomaterial (nPANI) is getting popular in many industrial fields due to its conductivity and stability. The fate and effect of nPANI in the environment is of paramount importance towards its technological applications. In this work, the cytotoxicity of nPANI, which was prepared by rapid surface polymerization, was studied on rat celiac macrophages. Cell viability of macrophages treated with various concentrations of nPANI and different periods ranging from 24 to 72 hours was tested by a MTT assay. Damages of nPANI to structures of macrophages were evaluated according to the exposure level of cellular reactive oxygen species (ROS) and change of mitochondrial membrane potential (MMP). We observed no significant effects of nPANI on the survival, ROS level and MMP loss of macrophages at concentrations up to 1 µg/ml. However, higher dose of nPANI (10 µg/ml or above) induced cell death, changes of ROS level and MMP. In addition, an increase in the expression level of caspase-3 protein and its activated form was detected in a Western blot assay under the high dose exposure of nPANI. All together, our experimental results suggest that the hazardous potential of nPANI on macrophages is time- and dose-dependent and high dose of nPANI can induce cell apoptosis through caspase-3 mediated pathway.

Keratin K18 increases cystic fibrosis transmembrane conductance regulator (CFTR) surface expression by binding to its C-terminal hydrophobic patch.

BACKGROUND: CFTR function is tightly regulated by many interacting proteins. RESULTS: Intermediate filament protein keratin 18 increases the cell surface expression of CFTR by interacting with the C-terminal hydrophobic patch of CFTR. CONCLUSION: K18 controls the function of CFTR. SIGNIFICANCE: These findings offer novel insights into the regulation of CFTR and suggest that K18 and its dimerization partner, K8, may be modifier genes in cystic fibrosis. Malfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) leads to cystic fibrosis, but the regulation of CFTR is not fully understood. Here, we identified the intermediate filament protein keratin K18 (K18) as a CFTR-binding protein by various approaches. We mapped a highly conserved "hydrophobic patch" ((1413)FLVI(1416)) in the CFTR C-terminus, known to determine plasmalemmal CFTR stability, as the K18-binding site. On the other hand, the C-terminal tail of K18 was found to be a critical determinant for binding CFTR. Overexpression of K18 in cells robustly increased the surface expression of wild-type CFTR, whereas depletion of K18 through RNA interference specifically diminished it. K18 binding increased the surface expression of CFTR by accelerating its apical recycling rate without altering CFTR biosynthesis, maturation, or internalization. Importantly, CFTR surface expression was markedly reduced in duodenal and gallbladder epithelia of K18(-/-) mice. Taken together, our results suggest that K18 increases the cell surface expression of CFTR by interacting with the CFTR C-terminal hydrophobic patch. These findings offer novel insights into the regulation of CFTR and suggest that K18 and its dimerization partner, K8, may be modifier genes in cystic fibrosis.

Mechanosensitive gating of CFTR.

Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion and intracellular ligand-gated channel associated with cystic fibrosis, a lethal genetic disorder common among Caucasians. Here we show that CFTR is robustly activated by membrane stretch induced by negative pressures as small as 5 mmHg at the single-channel, cellular and tissue levels. Stretch increased the product of the number of channels present and probability of being open (NPo), and also increased the unitary conductance of CFTR in cell-attached membrane patches. CFTR stretch-mediated activation appears to be an intrinsic property independent of cytosolic factors and kinase signalling. CFTR stretch-mediated activation resulted in chloride transport in Calu-3 human airway epithelial cells and mouse intestinal tissues. Our study has revealed an unexpected function of CFTR in mechanosensing, in addition to its roles as a ligand-gated anion channel and a regulator of other membrane transporters, demonstrating for the first time a mechanosensitive anion channel with a clearly defined molecular identity. Given that CFTR is often found in mechanically dynamic environments, its mechanosensitivity has important physiological implications in epithelial ion transport and cell volume regulation in vivo.