Methylation of Septin9, SRSF1, and PAX8 in Early Screening of Colorectal Cancer in the Population Undergoing Physical Examinations.

BACKGROUND: The aim was to investigate the value of blood Septin9, SRSF1, and PAX8 gene methylation detection techniques in early screening of colorectal cancer (CRC). METHODS: A prospective cohort study enrolled 3,000 participants undergoing routine physical examination at Shizong County People's Hospital Health Management Center from December 2021 through November 2022, including 1,512 males and 1,488 females, ranging in age from 20 to 90 years, with a median age of 49 years. Fresh blood samples were collected and tested for Septin9, SRSF1, and PAX8 gene methylation. Positive or negative results were reported. Colonoscopy was recommended for positive results and telephone follow-up for negative results. A chi-squared test analyzed the positive rate of initial screening, colonoscopy compliance, and the detection rate of colorectal lesions. Finally, combined with the follow-up data, the screening effect of Septin9, SRSF1, and PAX8 methylation detection on CRC was evaluated. RESULTS: Among 3,000 cases, 215 cases were preliminarily positive, with a positive rate of 7.1% (215/3,000). The positive rate of Septin9 gene methylation was the highest (6%, 180/3000), followed by SRSF1 (4.1%, 124/3000) and PAX8 (3.6%, 108/3000). The sensitivity of combined detection of Septin9, SRSF1, and PAX8 methylation in the diagnosis of CRC was higher than that of the three alone, and the specificity, positive predictive value, and negative predictive value of combined detection were higher than that of the single detection of blood Septin9, SRSF1, and PAX8 DNA methylation. In addition, the positive rate of initial screening increased with age (χ2 = 32.135, p < 0.001). A total of 150 cases underwent further colonoscopy, and the colonoscopy compliance rate was 69.8% (150/215). Among 150 cases who completed colonoscopy, 5 cases of CRC (3.4%), 25 cases of advanced adenoma (16.0%), 78 cases of non-advanced adenoma (52.0%), and 24 cases of non-adenomatous polyps (22.7%) were detected. The positive predictive value of Septin9, SRSF1, and PAX8 methylation was 94% (141/150) for all colorectal lesions, and 70.0% (105/150) for colorectal cancer and precancerous lesions. CONCLUSIONS: Blood Septin9, SRSF1, and PAX8 gene methylation detection, combined with colonoscopy, can effectively detect colorectal cancer and precancerous lesions. This strategy may be an effective way to carry out large-scale colorectal cancer screening in the general risk population. Combined detection of the three genes can improve the detection rate of colorectal cancer, but Septin9 methylation is the most sensitive, which can be used for screening and efficacy evaluation of CRC.

Controlled Genetic Encoding of Unnatural Amino Acids in a Protein Nanopore.

Conventional protein engineering methods for modifying protein nanopores are typically limited to 20 natural amino acids, which restrict the diversity of the nanopores in structure and function. To enrich the chemical environment inside the nanopore, we employed the genetic code expansion (GCE) technique to site-specifically incorporate the unnatural amino acid (UAA) into the sensing region of aerolysin nanopores. This approach leveraged the efficient pyrrolysine-based aminoacyl-tRNA synthetase-tRNA pair for a high yield of pore-forming protein. Both molecular dynamics (MD) simulations and single-molecule sensing experiments demonstrated that the conformation of UAA residues provided a favorable geometric orientation for the interactions of target molecules and the pore. This rationally designed chemical environment enabled the direct discrimination of multiple peptides containing hydrophobic amino acids. Our work provides a new framework for endowing nanopores with unique sensing properties that are difficult to achieve using classical protein engineering approaches.

Bioorthogonally Activatable Base Editing for On-Demand Pyroptosis.

Pyroptosis is an inflammatory cell death form triggered by protease-mediated truncation and release of the N-terminal pore-forming domain of the gasdermin (GSDM) family proteins in various cell types. We report a Bioorthogonally ACtivatable Base editor (BaseBAC) for in situ and on-demand initiation of cell-type-specific pyroptosis. We first made the enzymatic activity of a cytosine base editor (CBE) switchable by establishing a bioorthogonal blockage on the PAM-interacting residue to control its DNA-binding ability. The resulting BaseBAC allowed in situ control of base editing on the GSDME gene that switched to the truncated expression of its N-terminal domain to activate pyroptosis. BaseBAC offers a general method for on-demand awakening of functional domains of self-inhibiting proteins and the corresponding cellular processes with high specificity in living systems.

Cationic Lipid-based Intracellular Delivery of Bacterial Effectors for Rewiring Malignant Cell Signaling.

The abundance of bacterial effectors have inspired us to explore their potential in rewiring malignant cell signaling. Their incapability for entering cells, however, hinders such application. Herein we developed a cationic lipid-based high throughput library screening platform for effective intracellular delivery of bacterial effectors. As the misregulated MAPK signaling is a hallmark of many types of cancer, we turned to the Shigella effector OspF which irreversibly inactivates ERK, the terminal component of MAPK cascade. We created a function-based screening assay to obtain AMPA-O16B lipid nanoparticles for effective OspF intracellular delivery, which inhibited the malignant MAPK signaling and tumor growth in vitro and in vivo. Furthermore, the optimized lipid nanoparticle formulation can deliver OspF to modulate the immunosuppressive responses in macrophages. Our work is a general strategy to explore the therapeutic potentials of naturally evolved bacterial effectors.

Quinone and nitrofurantoin redox cycling by recombinant cytochrome b5 reductase.

NADH cytochrome b5 reductase mediates electron transfer from NADH to cytochrome b5 utilizing flavin adenine dinucleotide as a redox cofactor. Reduced cytochrome b5 is an important cofactor in many metabolic reactions including cytochrome P450-mediated xenobiotic metabolism, steroid biosynthesis and fatty acid metabolism, hemoglobin reduction, and methionine and plasmalogen synthesis. Using recombinant human enzyme, we discovered that cytochrome b5 reductase mediates redox cycling of a variety of quinones generating superoxide anion, hydrogen peroxide, and, in the presence of transition metals, hydroxyl radicals. Redox cycling activity was oxygen-dependent and preferentially utilized NADH as a co-substrate; NADH was 5-10 times more active than NADPH in supporting redox cycling. Redox cycling activity was greatest for 9,10-phenanthrenequinone and 1,2-naphthoquinone, followed by 1,4-naphthoquinone and 2-methyl-1,4-naphthoquinone (menadione), nitrofurantoin and 2-hydroxyestradiol. Using menadione as the substrate, quinone redox cycling was found to inhibit reduction of cytochrome b5 by cytochrome b5 reductase, as measured by heme spectral changes in cytochrome b5. Under anaerobic conditions where redox cycling is inhibited, menadione had no effect on the reduction of cytochrome b5. Chemical redox cycling by cytochrome b5 reductase may be important in generating cytotoxic reactive oxygen species in target tissues. This activity, together with the inhibition of cytochrome b5 reduction by redox-active chemicals and consequent deficiencies in available cellular cytochrome b5, are likely to contribute to tissue injury following exposure to quinones and related redox active chemicals.

The NLRP3 Inflammasome: An Important Driver of Neuroinflammation in Hemorrhagic Stroke.

Hemorrhagic stroke is a devastating clinical event with no effective medical treatment. Neuroinflammation, which follows a hemorrhagic stroke, is an important element that involves both acute brain injury and subsequent brain rehabilitation. Therefore, delineating the key inflammatory mediators and deciphering their pathophysiological roles in hemorrhagic strokes is of great importance in the development of novel therapeutic targets for this disease. The NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome is a multi-protein complex that is localized within the cytoplasm. This NOD-like receptor orchestrates innate immune responses to pathogenic organisms and cell stress through the activation of caspase-1 and the maturation of the proinflammatory cytokines such as interleukin-1ß (IL-1ß) and IL-18. Mounting evidence has demonstrated that when the NLRP3 inflammasome is activated, it exerts harmful effects on brain tissue after a hemorrhagic stroke. This review article summarizes the current knowledge regarding the role and the underlying mechanisms of the NLRP3 inflammasome in the pathophysiological processes of hemorrhagic strokes. A better understanding of the function and regulation of the NLRP3 inflammasome in hemorrhagic strokes will provide clues for devising novel therapeutic strategies to fight this disease.

Diacetyl/l-Xylulose Reductase Mediates Chemical Redox Cycling in Lung Epithelial Cells.

Reactive carbonyls such as diacetyl (2,3-butanedione) and 2,3-pentanedione in tobacco and many food and consumer products are known to cause severe respiratory diseases. Many of these chemicals are detoxified by carbonyl reductases in the lung, in particular, dicarbonyl/l-xylulose reductase (DCXR), a multifunctional enzyme important in glucose metabolism. DCXR is a member of the short-chain dehydrogenase/reductase (SDR) superfamily. Using recombinant human enzyme, we discovered that DCXR mediates redox cycling of a variety of quinones generating superoxide anion, hydrogen peroxide, and, in the presence of transition metals, hydroxyl radicals. Redox cycling activity preferentially utilized NADH as a cosubstrate and was greatest for 9,10-phenanthrenequinone and 1,2-naphthoquinone, followed by 1,4-naphthoquinone and 2-methyl-1,4-naphthoquinone (menadione). Using 9,10-phenanthrenequinone as the substrate, quinone redox cycling was found to inhibit DCXR reduction of l-xylulose and diacetyl. Competitive inhibition of enzyme activity by the quinone was observed with respect to diacetyl (Ki = 190 µM) and l-xylulose (Ki = 940 µM). Abundant DCXR activity was identified in A549 lung epithelial cells when diacetyl was used as a substrate. Quinones inhibited reduction of this dicarbonyl, causing an accumulation of diacetyl in the cells and culture medium and a decrease in acetoin, the reduced product of diacetyl. The identification of DCXR as an enzyme activity mediating chemical redox cycling suggests that it may be important in generating cytotoxic reactive oxygen species in the lung. These activities, together with the inhibition of dicarbonyl/l-xylulose metabolism by redox-active chemicals, as well as consequent deficiencies in pentose metabolism, are likely to contribute to lung injury following exposure to dicarbonyls and quinones.

Early secreted antigen ESAT-6 of Mycobacterium Tuberculosis promotes apoptosis of macrophages via targeting the microRNA155-SOCS1 interaction.

BACKGROUND: The early secreted antigenic target 6-kDa protein (ESAT-6) of Mycobacterium tuberculosis (Mtb) not only acts as a key player for virulence but also exhibits a strong immunotherapeutic potential against Mtb. However, little is known about the molecular basis for its potential in immunotherapy. The present study was designed to unravel the role of miRNA-155 in ESAT-6-mediated enhancement of host immunity and apoptosis in macrophages. METHODS: Lentivirus-mediated miR-155 sponge and miR-155 and SOCS1 overexpression vectors were developed in macrophages. TLR2- or p65-specific siRNA knockdown was employed to silence TLR2 or p65. Quantitative polymerase chain reaction and western blotting analyses were performed to determine mRNA and protein expression levels, respectively. Macrophage apoptosis was analyzed by flow cytometry. RESULTS: ESAT-6 significantly increased miR-155 expression, which was dependent on TLR2/NF-κB activation in macrophages. Induced expression of miRNA-155 was required for the ESAT-6-mediated protective immune response and macrophage apoptosis. ESAT-6 promoted macrophage apoptosis by targeting the miR-155-SOCS1 pathway. The differential expression levels of TLR2, BIC, and SOCS1 were involved in regulating the immune response in human peripheral blood mononuclear cells of patients with active tuberculosis (TB) and latent TB (LTB). CONCLUSION: ESAT-6 promotes apoptosis of macrophages via targeting the miRNA155-SOCS1 interaction.

Sulfa drugs inhibit sepiapterin reduction and chemical redox cycling by sepiapterin reductase.

Sepiapterin reductase (SPR) catalyzes the reduction of sepiapterin to dihydrobiopterin (BH2), the precursor for tetrahydrobiopterin (BH4), a cofactor critical for nitric oxide biosynthesis and alkylglycerol and aromatic amino acid metabolism. SPR also mediates chemical redox cycling, catalyzing one-electron reduction of redox-active chemicals, including quinones and bipyridinium herbicides (e.g., menadione, 9,10-phenanthrenequinone, and diquat); rapid reaction of the reduced radicals with molecular oxygen generates reactive oxygen species (ROS). Using recombinant human SPR, sulfonamide- and sulfonylurea-based sulfa drugs were found to be potent noncompetitive inhibitors of both sepiapterin reduction and redox cycling. The most potent inhibitors of sepiapterin reduction (IC50s = 31-180 nM) were sulfasalazine, sulfathiazole, sulfapyridine, sulfamethoxazole, and chlorpropamide. Higher concentrations of the sulfa drugs (IC50s = 0.37-19.4 µM) were required to inhibit redox cycling, presumably because of distinct mechanisms of sepiapterin reduction and redox cycling. In PC12 cells, which generate catecholamine and monoamine neurotransmitters via BH4-dependent amino acid hydroxylases, sulfa drugs inhibited both BH2/BH4 biosynthesis and redox cycling mediated by SPR. Inhibition of BH2/BH4 resulted in decreased production of dopamine and dopamine metabolites, 3,4-dihydroxyphenylacetic acid and homovanillic acid, and 5-hydroxytryptamine. Sulfathiazole (200 µM) markedly suppressed neurotransmitter production, an effect reversed by BH4. These data suggest that SPR and BH4-dependent enzymes, are "off-targets" of sulfa drugs, which may underlie their untoward effects. The ability of the sulfa drugs to inhibit redox cycling may ameliorate ROS-mediated toxicity generated by redox active drugs and chemicals, contributing to their anti-inflammatory activity.

Sepiapterin reductase mediates chemical redox cycling in lung epithelial cells.

In the lung, chemical redox cycling generates highly toxic reactive oxygen species that can cause alveolar inflammation and damage to the epithelium, as well as fibrosis. In this study, we identified a cytosolic NADPH-dependent redox cycling activity in mouse lung epithelial cells as sepiapterin reductase (SPR), an enzyme important for the biosynthesis of tetrahydrobiopterin. Human SPR was cloned and characterized. In addition to reducing sepiapterin, SPR mediated chemical redox cycling of bipyridinium herbicides and various quinones; this activity was greatest for 1,2-naphthoquinone followed by 9,10-phenanthrenequinone, 1,4-naphthoquinone, menadione, and 2,3-dimethyl-1,4-naphthoquinone. Whereas redox cycling chemicals inhibited sepiapterin reduction, sepiapterin had no effect on redox cycling. Additionally, inhibitors such as dicoumarol, N-acetylserotonin, and indomethacin blocked sepiapterin reduction, with no effect on redox cycling. Non-redox cycling quinones, including benzoquinone and phenylquinone, were competitive inhibitors of sepiapterin reduction but noncompetitive redox cycling inhibitors. Site-directed mutagenesis of the SPR C-terminal substrate-binding site (D257H) completely inhibited sepiapterin reduction but had minimal effects on redox cycling. These data indicate that SPR-mediated reduction of sepiapterin and redox cycling occur by distinct mechanisms. The identification of SPR as a key enzyme mediating chemical redox cycling suggests that it may be important in generating cytotoxic reactive oxygen species in the lung. This activity, together with inhibition of sepiapterin reduction by redox-active chemicals and consequent deficiencies in tetrahydrobiopterin, may contribute to tissue injury.

Rapid detection of hepatitis B virus variants associated with lamivudine and adefovir resistance by multiplex ligation-dependent probe amplification combined with real-time PCR.

Drug-resistant mutations of hepatitis B virus (HBV) are the major obstacles to successful therapy for chronic hepatitis B infection. Although there are many methods for detecting the antiviral drug-resistant mutations of HBV, their applications are restricted because of their shortcomings, such as low sensitivity, the time required, and the high cost. For this study, a multiplex ligation-dependent probe real-time PCR (MLP-RT-PCR) method was developed to simultaneously detect lamivudine (LAM)- and adefovir (ADV)-resistant HBV mutants (those with the mutations rtM204V/I, rtA181V/T, and rtN236T). The new method combined the high-throughput nature of multiplex ligation-dependent probe amplification (MLPA) with the rapid and sensitive detection of real-time PCR. In this report, MLP-RT-PCR was evaluated by detecting drug-resistant mutants in 116 patients with chronic hepatitis B infection. By MLP-RT-PCR analysis, LAM-resistant mutations were detected in 41 patients (35.3%), ADV-resistant mutations were detected in 17 patients (14.7%), and LAM- and-ADV-resistant mutations were detected in 5 patients (4.3%). Based on the results of MLP-RT-PCR, the mutations rtM204V, rtM204I, rtA181T, rtA181V, and rtN236T were 95.7% (111/116 patients), 98.3% (114/116 patients), 99.1% (115/116 patients), 98.3% (114/116 patients), and 99.1% (115/116 patients) concordant, respectively, with those of direct sequencing. The MLP-RT-PCR assay was more sensitive than direct sequencing for detecting mutations with low frequencies. Four samples containing the low-frequency (<10%) mutants were identified by MLP-RT-PCR and further confirmed by clonal sequencing. MLP-RT-PCR is a rapid and sensitive method that enables the detection of multidrug-resistant HBV mutations in clinical practice.

W-WaveNet: A multi-site water quality prediction model incorporating adaptive graph convolution and CNN-LSTM.

Water quality prediction is of great significance in pollution control, prevention, and management. Deep learning models have been applied to water quality prediction in many recent studies. However, most existing deep learning models for water quality prediction are used for single-site data, only considering the time dependency of water quality data and ignoring the spatial correlation among multi-sites. This research defines and analyzes the non-aligned spatial correlations that exist in multi-site water quality data. Then deploy spatial-temporal graph convolution to process water quality data, which takes into account both the temporal and spatial correlation of multi-site water quality data. A multi-site water pollution prediction method called W-WaveNet is proposed that integrates adaptive graph convolution and Convolutional Neural Network, Long Short-Term Memory (CNN-LSTM). It integrates temporal and spatial models by interleaved stacking. Theoretical analysis shows that the method can deal with non-aligned spatial correlations in different time spans, which is suitable for water quality data processing. The model validates water quality data generated on two real river sections that have multiple sites. The experimental results were compared with the results of Support Vector Regression, CNN-LSTM, and Spatial-Temporal Graph Convolutional Networks (STGCN). It shows that when W-WaveNet predicts water quality over two river sections, the average Mean Absolute Error is 0.264, which is 45.2% lower than the commonly used CNN-LSTM model and 23.8% lower than the STGCN. The comparison experiments also demonstrate that W-WaveNet has a more stable performance in predicting longer sequences.

Individual variation in and lateral asymmetry of mouse epididymal draining lymph nodes.

PROBLEM: Draining lymph nodes (LNs) are pivotal sites for maintaining tolerance to self-antigens as well as eliciting immune responses to exogenous antigens. The epididymis is a male reproductive organ with a unique local immune environment. Although mice are the most commonly used laboratory animals for immunology research, there are no detailed descriptions of the anatomical location and function of LNs that drain the epididymis. METHOD OF STUDY: Evans blue labeling was utilized to explore lymphatic drainage of the epididymis in eight- to ten-week-old male C57BL/6 mice. We confirmed the lymphatic drainage of the epididymis in mice using the objective technique of carboxyfluorescein succinimidyl ester (CFSE)-labeled cells. RESULTS: By combined Evans blue labeling and fluorescent labeling, we found that 1) the patterns of epididymal LN drainage are highly heterogeneous between individual mice; 2) the leftside LNs participate in drainage more frequently than the right-side LNs; and 3) epididymal lymphatic drainage bypasses both the paraaortic and renal LNs in some mice. CONCLUSIONS: These data highlighted the need to consider the individual variation in and lateral asymmetry of draining LNs when characterizing the regional immunology of the mouse epididymis.

Effects of different light intensity on the growth of tomato seedlings in a plant factory.

Light-emitting diodes (LEDs) were the best artificial light source for plant factories. Red light-emitting diodes (LEDs, R) and blue light-emitting diodes (LEDs, B) were used to obtain different light intensities of uniform spectra, and the greenhouse environment was considered as a comparison. The results showed that root dry weight, shoot dry weight and stem diameter were superior in plant growth under 240 µmolm-2s-1, additionally, the Dixon Quality Index (DQI) was also best. Under 240 µmolm-2s-1, the net photosynthesis rate (Pn) was consistent with the greenhouse's treatment, superior to other experimental groups. The results implied that the PPFD was more suitable for the cultivation of tomato seedlings under the condition of 240 µmolm-2s-1, and can replace the greenhouse conditions so as to save energy and reduce emissions.

Effects of Light Intensity on Growth and Quality of Lettuce and Spinach Cultivars in a Plant Factory.

The decreased quality of leafy vegetables and tipburn caused by inappropriate light intensity are serious problems faced in plant factories, greatly reducing the economic benefits. The purpose of this study was to comprehensively understand the impact of light intensity on the growth and quality of different crops and to develop precise lighting schemes for specific cultivars. Two lettuce (Lactuca sativa L.) cultivars-Crunchy and Deangelia-and one spinach (Spinacia oleracea L.) cultivar-Shawen-were grown in a plant factory using a light-emitting diode (LED) under intensities of 300, 240, 180, and 120 µmol m-2 s-1, respectively. Cultivation in a solar greenhouse using only natural light (NL) served as the control. The plant height, number of leaves, and leaf width exhibited the highest values under a light intensity of 300 µmol m-2 s-1 for Crunchy. The plant width and leaf length of Deangelia exhibited the smallest values under a light intensity of 300 µmol m-2 s-1. The fresh weight of shoot and root, soluble sugar, soluble protein, and ascorbic acid contents in the three cultivars increased with the increasing light intensity. However, tipburn was observed in Crunchy under 300 µmol m-2 s-1 light intensity, and in Deangelia under both 300 and 240 µmol m-2 s-1 light intensities. Shawen spinach exhibited leaf curling under all four light intensities. The light intensities of 240 and 180 µmol m-2 s-1 were observed to be the most optimum for Crunchy and Deangelia (semi-heading lettuce variety), respectively, which would exhibit relative balance growth and morphogenesis. The lack of healthy leaves in Shawen spinach under all light intensities indicated the need to comprehensively optimize cultivation for Shawen in plant factories to achieve successful cultivation. The results indicated that light intensity is an important factor and should be optimized for specific crop species and cultivars to achieve healthy growth in plant factories.

Fabrication of an electrochemical platform based on the self-assembly of graphene oxide-multiwall carbon nanotube nanocomposite and horseradish peroxidase: direct electrochemistry and electrocatalysis.

A novel hybrid nanomaterial (GO-MWNTs) was explored based on the self-assembly of multiwall carbon nanotubes (MWNTs) and graphene oxide (GO). Compared with pristine MWNTs, such a nanocomposite could be well dispersed in aqueous solution and exhibit a negative charge. Driven by the electrostatic interaction, positively charged horseradish peroxidase (HRP) could then be immobilized onto GO-MWNTs at the surface of a glassy carbon (GC) electrode to form a HRP/GO-MWNT/GC electrode under mild conditions. TEM was used to characterize the morphology of the GO-MWNT nanocomposite. UV-vis and FTIR spectra suggested that HRP was immobilized onto the hybrid matrix without denaturation. Furthermore, the immobilized HRP showed enhanced direct electron transfer for the HRP-Fe(III)/Fe(II) redox center. Based on the direct electron transfer of the immobilized HRP, the HRP/GO-MWNT/GC electrode exhibited excellent electrocatalytic behavior to the reduction of H(2)O(2) and NaNO(2), respectively. Therefore, GO-MWNTs could provide a novel and efficient platform for the immobilization and biosensing of redox enzymes, and thus may find wide potential applications in the fabrication of biosensors, biomedical devices, and bioelectronics.

Chemical engineering of bacterial effectors for regulating cell signaling and responses.

Bacteria have evolved a variety of effector proteins to facilitate their survival and proliferation within the host environment. Continuous competition at the host-pathogen interface has empowered these effectors with unique mechanism and high specificity toward their host targets. The rich repertoire of bacterial effectors has thus provided us an attractive toolkit for investigating various cellular processes, such as signal transductions. With recent advances in protein chemistry and engineering, we now have the capability for on-demand control of protein activity with high precision. Herein, we review the development of chemically engineered bacterial effectors to control kinase-mediated signal transductions, inhibit protein translation, and direct genetic editing within host cells. We also highlight future opportunities for harnessing diverse prokaryotic effectors as powerful tools for mechanistic investigation and therapeutic intervention of eukaryotic systems.

Efficacy of Cigu Xiaozhi pill on non-alcoholic steatohepatitis-associated lipoapoptosis through stress-activated c-Jun N-terminal kinase signalling pathway.

OBJECTIVE: To investigate the efficacy of Cigu Xiaozhi pill (, CGXZ) on non-alcoholic steatohepatitis (NASH)-associated lipoapoptosis through the stress-activated c-Jun N-terminal kinase (JNK)/ stress-activated protein kinase signalling pathway. METHODS: Sixty male Sprague-Dawley rats were randomly divided into the following groups (10rats each): blank control, model, low-dose CGXZ, medium-dose CGXZ, high-dose CGXZ, and positive control (treated with SP600125, a JNK inhibitor). The NASH model was established and the histomorphological characteristics of haematoxylin and eosin-stained liver tissues were examined under a light microscope. Cell apoptosis in liver tissues was assessed via terminal deoxynucleotidyl transferase dUTP nick-end labelling assay. In addition, the mRNA and protein expression levels of p-JNK, p-c-Jun, caspase-8, Fas, and Fas-L were determined via fluorescence-based quantitative real-time PCR, immunohistochemical and Western blot assays. RESULTS: Histopathological examination of the liver showed that the model rats had moderate-to-severe steatosis with infiltration of inflammatory cells as well as significantly higher expression levels of the p-JNK, p-c-Jun, caspase-8, Fas, and Fas-L proteins, compared with those in the blank control group (P < 0.01). Hepatic lobules of the rats in the treatment groups showed significantly reduced vacuolar degeneration and steatosis as well as alleviated inflammatory cell infiltration. The high and medium-dose CGXZ groups exhibited significantly lower mRNA and protein expression levels of p-JNK, p-c-Jun, caspase-8, Fas, and Fas-L, compared with those in the model group (P < 0.05 or P < 0.01). CONCLUSION: CGXZ pill inhibited the onset of hepatocyte apoptosis by regulating the expression of p-JNK, p-c-Jun, caspase-8, Fas, and Fas-L, thereby exerting therapeutic effects against NASH.

[Cigu Xiaozhi pills's influence on lipid peroxidation and TNF-alpha expression in liver tissues of rats with nonalcoholic steatohepatitis].

OBJECTIVE: To investigate the effect of Cigu Xiaozhi pills on expression of tumor necrosis factor alpha (TNF-alpha) in rat with nonalcoholic steatoheptatitis (NASH). METHOD: Fifty male SD rats were divided randomly into five groups: the normal group (n = 10), the model group (n = 10), the high dosage group (n = 10) and the little dosage group of the treatment (n = 10), Dongbao Gantai group (n = 10). Rats in normal control group were fed with standard diet. Rats in other four groups were established models of nonalcoholic steatohepatitis and were treated simultaneously with traditional Chinese medicine and positive medicine. Histopathological changes in the liver were observed. The serum ALT, AST, TG, TC and hepatic MDA, SOD, GPX were detected histologically. The expression of TNF-alpha in the liver was determined using the immunohistochemical technique and RT-PCR. RESULT: In model group, extensive adipose degeneration and inflammatory cell infiltration were found in the liver. In treatment groups steatohepatitis were markedly alleviated. Compared with model group, TG, TC, ALT, AST of the serum and MDA of liver tissue were reduced significantly, and SOD, GPX were increased significantly in treatment groups and positive control group (P < 0.05). TNF-alpha was not expressed almost in normal rat liver and was expressed highly in model rat liver. Compared with model group, the TNF-alpha mRNA and protein expression were significantly lower in liver of treatment groups and positive control group (P < 0.05). And the effects of high dosage group surpass than those of Dongbao Gantai group (P < 0.05). CONCLUSION: TNF-alpha plays an important role in NASH pathogenesis. Cigu Xiaozhi pills can effectively treat experimental nonalcoholic steatohepatitis in rats, and its mechanism may be associated with ameliorating hepatocellular steatosis, removing the free radicals and enhancing the capability of anti-oxidation and anti-inflammatory.

Noni (Morinda citrifolia L.) wine prevents the oxidative stress and obesity in mice induced by high-fat diet.

Noni (Morinda citrifolia L.) is rich in polyphenols, flavonoids, terpenoids, and iridoids. However, its bad taste and smell make noni fruit unsuitable for consumption. After fermentation, noni wine becomes free from the undesirable smell. Nevertheless, it is still unclear whether processed noni could retain its original nutrients and effects. Therefore, we conducted a series of evaluations on the nutritional composition and efficacy of noni wine. Our results showed that the polyphenol, flavonoid, and vitamin C contents in noni wine were 558.80, 234.42, and 0.30 mg/L, respectively. Our animal experiments showed that 40 ml kg-1  day-1 noni wine could reduce bodyweight, as well as the levels of body fat, serum triglycerides, total cholesterol, and low-density lipoprotein, while it simultaneously increased the amount of energy expenditure and activity, and improved the systemic antioxidant capacity in mice following a high-fat diet. The results of the gene expression and western blot analyses showed that 40 ml kg-1  day-1 noni wine could regulate the Nrf2 pathway and improve the antioxidant enzyme gene expression in mice maintained on a high-fat diet, thereby improving body lipid metabolism, reducing fatty acid synthesis, and promoting fatty acid ß-oxidation. Our study indicated that drinking 40 ml kg-1  day-1 noni wine could effectively prevent high-fat diet-induced oxidative stress and obesity in mice. PRACTICAL APPLICATIONS: Noni fruit is rich in nutrients but its bad smell and hardship of processing make its commercialization difficult. Previous studies mainly focused on fresh noni juice and its primary processed products, while few noni products, of poor taste and low quality, are available in the market. Therefore, the fruit wine with both the nutritive values and the special flavor of noni has broad market prospects. Our work provides a valuable reference for the commercialization of noni wine.