[Effect of Uremic Clearance Granules on improvement of chronic kidney disease in rats based on microbiome-metabolomics and its mechanism].

This research aimed to study the effect of Uremic Clearance Granules on chronic kidney disease in SD rats by using the methods of microbial functional genomics combined with metabolomics, and to preliminarily explore its mechanism. The SD rat model of chronic kidney disease was established by the adenine-induced method. After the model was successfully induced, the animals were randomly divided into a negative control group, a Uremic Clearance Granule treatment group, and a normal control group, with 8 rats in each group. After 4 weeks of administration, animal feces and serum were collected, and 16S rDNA sequencing technology was used to analyze the abundance, diversity, and function prediction of intestinal microorganisms. Liquid chromatography-mass spectrometry(LC-MS) technology was used to perform high-throughput sequencing to detect animal serum metabolites. The MetPA database was used to screen out potential biomarkers of chronic kidney disease in rats and conduct the enrichment analysis of metabolic pathways. Spearman's method was used to analyze the correlation between the two omics. The results showed that Uremic Clearance Granules effectively improved the body weight loss and renal function-related biochemical and appearance indicators in rats with chronic kidney disease. The results of 16S rDNA sequencing showed that Uremic Clearance Granules regulated the diversity and composition of the intestinal flora in rats with chronic kidney disease. The changes in the intestinal flora affected functional metabolic pathways such as amino acid biosynthesis and metabolism, lipid metabolism, and carbohydrate metabolism. The results of LC-MS showed that as compared with the negative control group, 15 metabolites were reversed in the Uremic Clearance Granule treatment group, among which 11 potential marker metabolites were significantly up-regulated and 4 potential marker metabolites were significantly down-regulated. Five amino acid metabolic pathways were mainly involved, which were significantly correlated with changes in the intestinal flora. Therefore, Uremic Clearance Granules can improve the renal function of rats with chronic kidney disease, and the mechanism may be related to its effect on the amino acid metabolism pathway by regulating the intestinal flora.

LMWP (S3-3) from the Larvae of Musca domestica Alleviate D-IBS by Adjusting the Gut Microbiota.

Diarrhea-based Irritable Bowel Syndrome (D-IBS) and diarrhea are both associated with ecological imbalance of the gut microbiota. Low Molecular Weight Peptides (LMWP) from the larvae of Musca domestica have been shown to be effective in the treatment of diarrhea and regulation of gut microbiota. Meanwhile, the single polypeptide S3-3 was successfully isolated and identified from LMWP in our previous studies. It remains unclear exactly whether and how LMWP (S3-3) alleviate D-IBS through regulating gut microbiota. We evaluated the gut microbiota and pharmacology to determine the regulation of gut microbiota structure and the alleviating effect on D-IBS through LMWP (S3-3). The rates of loose stools, abdominal withdrawal reflex (AWR) and intestinal tract motility results revealed that LMWP (S3-3) from the larvae of Musca domestica had a regulating effect against diarrhea, visceral hypersensitivity and gastrointestinal (GI) dysfunction in D-IBS model mice. Additionally, 16S rRNA gene sequencing was utilized to examine the gut microbiota, which suggests that LMWP induce structural changes in the gut microbiota and alter the levels of the following gut microbiota: Bacteroidetes, Proteobacteria and Verrucomicrobia. LMWP putatively functioned through regulating 5-HT, SERT, 5-HT2AR, 5-HT3AR and 5-HT4R according to the results of ELISA, qRT-PCR and IHC. The findings of this study will contribute to further understanding how LMWP (S3-3) attenuate the effects of D-IBS on diarrhea, visceral hypersensitivity and GI dysfunction.

Effects of Chang-Kang-Fang Formula on the Microbiota-Gut-Brain Axis in Rats With Irritable Bowel Syndrome.

Chang-Kang-Fang formula (CKF), a multi-herb traditional Chinese medicine, has been used in clinical settings to treat irritable bowel syndrome (IBS). Recent studies show that 5.0 g/kg/d CKF can alleviate the symptoms of IBS rats by modulating the brain-gut axis through the production of brain-gut peptides (BGPs), thus relieving pain, and reversing the effects of intestinal propulsion disorders. However, the exact mechanisms underlying the therapeutic effects of CKF in IBS remain unclear. The microbiota-gut-brain axis (MGBA) is central to the pathogenesis of IBS, regulating BGPs, depression-like behaviors, and gut microbiota. Given that CKF ameliorates IBS via the MGBA, we performed metabolomic analyses, evaluated the gut microbiota, and system pharmacology to elucidate the mechanisms of action of CKF. The results of intestinal tract motility, abdominal withdrawal reflex (AWR), sucrose preference test (SPT), and the forced swimming test (FST) showed that the male Sprague-Dawley rats subjected to chronic acute combining stress (CACS) for 22 days exhibited altered intestinal motility, visceral hypersensitivity, and depression-like behaviors. Treatment of IBS rats with CKF normalized dysfunctions of CACS-induced central and peripheral nervous system. CKF regulated BDNF and 5-HT levels in the colon and hippocampus as well as the expressions of the related BGP pathway genes. Moreover, the system pharmacology assays were used to assess the physiological targets involved in the action of CKF, with results suggesting that CKF putatively functioned through the 5-HT-PKA-CREB-BDNF pathway. LC-MS-based metabolomics identified the significantly altered 5-HT pathway-related metabolites in the CKF treatment group, and thus, the CKF-related signaling pathways were further examined. After pyrosequencing-based analysis of bacterial 16S rRNA (V3 + V4 region) using rat feces, the Lefse analysis of gut microbiota suggested that CKF treatment could induce structural changes in the gut microbiota, thereby regulating it by decreasing Clostridiales, and the F-B ratio while increasing the levels of Lactobacillus. Furthermore, the integrated analysis showed a correlation of CKF-associated microbes with metabolites. These findings showed that CKF effectively alleviated IBS, which was associated with the altered features of the metabolite profiles and the gut microbiota through a bidirectional communication along the microbiota-gut-brain axis.

Beneficial effect of simvastatin on human umbilical vein endothelial cells gap junctions induced by TNF-α.

Although simvastatin has been shown to inhibit vascular permeability, which might be amplified via gap junction intercellular communication (GJIC), the underlying mechanism of action remains unclear. In the present study, we investigated the effects and mechanisms of simvastatin on endothelial cells GJIC. Specifically, human umbilical vein endothelial cells (HUVECs) were stimulated with TNF-α (10 ng/mL) alone or in combination with simvastatin (5 µM), and their effects on vascular endothelial cell GJIC tested via the scrape loading/dye transfer (SL/DT) assay. Next, we performed immunofluorescence, real-time PCR and western blot assays to analyze expression of Cx37, Cx40 and Cx43 in HUVECs. Results showed that GJIC activity in HUVECs was markedly elevated in HUVECs treated with TNF-α in combination with simvastatin. In addition, simvastatin treatment significantly upregulated expression of Cx37 and Cx40 but downregulated Cx43 mRNAs and proteins. Taken together, these marked changes indicated that simvastatin exerts its regulatory effects on gap junction function by upregulating Cx37 and Cx40 and downregulating Cx43 expression.

[Preliminary study on effect of Phellinus igniarius ethanol extract on serum uric acid metabolism and gut microbiome in rats].

The aim of this paper was to investigate the effect of ethanol extract of Phellinus igniarius in lowering uric acid and changing the gut microbiome in hyperuricemia rats. A total of 36 SD rats were randomly divided into normal control group, model control group, positive drug control group, and high-dose, middle-dose and low-dose P. igniarius ethanol extract groups, with 6 rats in each group. Hyperuricemia rats were established by D-fructose combined with oteracil potassium(OAPS). One week later, the positive control group was given allopurinol 50 mg·kg~(-1) intragastrically, and P. igniarius ethanol extract groups were treated with 30, 60 and 90 mg·kg~(-1) drugs for 14 consecutive days. Body weight, blood glucose and serum uric acid(SUA) were monitored every week. After the model rats were administered with the ethanol extracts of P. igniarius by gavage for two weeks, the activities of creatinine, BUN, xanthine oxidase(XOD) and adenosine deaminase(ADA) were detected. The right kidney was taken to analyze the histological and morphological changes and the degree of damage to main organs of the extract of P. igniarius. The 16 S rDNA gene sequence technique was used to analyze the guts microbiota composition in feces. The results indicated that ethanol extract of P. igniarius could significantly lower the SUA level(P<0.01), while inhibiting the activities of XOD and ADA(P<0.05, P<0.01). Histological examination showed that the allopurine group showed slight renal tubular dilation and inflammatory cell infiltration compared with the normal group, with no significant difference between the P. igniarius ethanol extract groups and the normal group. The 16 S sequencing results showed that the composition of gut microbiota has changed in each group. Therefore, ethanol extracts of P. igniarius may reduce the level of SUA in rats by inhibiting the activities of XOD and ADA, with a certain effect on the composition of gut microbiota.

Colon tissue-accumulating mesoporous carbon nanoparticles loaded with Musca domestica cecropin for ulcerative colitis therapy.

Ulcerative colitis (UC) is a modern refractory disease with steadily increasing incidence worldwide that urgently requires effective and safe therapies. Therapeutic peptides delivered using nanocarriers have shown promising developments for the treatment of UC. We developed a novel colon-accumulating oral drug delivery nanoplatform consisting of Musca domestica cecropin (MDC) and mesoporous carbon nanoparticles (MCNs) and investigated its effects and mechanism of action for the treatment of UC. Methods: An optimized one-step soft templating method was developed to synthesize MCNs, into which MDC was loaded to fabricate MDC@MCNs. MCNs and MDC@MCNs were characterized by BET, XRD, and TEM. MDC and MDC@MCNs resistance to trypsin degradation was measured through Oxford cup antibacterial experiments using Salmonella typhimurium as the indicator. Uptake of MDC and MDC@MCNs by NCM460 cells was observed by fluorescence microscopy. The biocompatibility of MDC, MCNs, and MDC@MCNs was evaluated in three cell lines (NCM460, L02, and NIH3T3) and C57BL/6 mice. Dextran sulphate sodium was used to establish models of NCM460 cell injury and UC in mice. MTT assay, flow cytometry, and mitochondrial membrane potential assay were applied to determine the effects of MDC@MCNs on NCM460 cells injury. Additionally, a variety of biological methods such as H&E staining, TEM, ELISA, qPCR, Western blotting, and 16s rDNA sequencing were performed to explore the effects and underlying mechanism of MDC@MCN on UC in vivo. Colonic adhesion of MCNs was compared in normal and UC mice. The oral biodistributions of MDC and MDC@MCNs in the gastrointestinal tract of mice were also determined. Results: MDC@MCNs were successfully developed and exhibited excellent ability to resist destruction by trypsin and were taken up by NCM460 cells more readily than MDC. In vitro studies showed that MDC@MCNs better inhibited DSS-induced NCM460 cells damage with lower toxicity to L02 and NIH3T3 cells compared with MDC. In vivo results indicated that MDC@MCNs have good biocompatibility and significantly improved colonic injury in UC mice by effectively inhibiting inflammation and oxidative stress, maintaining colonic tight junctions, and regulating intestinal flora. Moreover, MDC@MCNs were strongly retained in the intestines, which was attributed to intestinal adhesion and aggregation of MCNs, serving as one of the important reasons for its enhanced efficacy after oral administration compared with MDC. Conclusion: MDC@MCNs alleviated DSS-induced UC by ameliorating colonic epithelial cells damage, inhibiting inflammation and oxidative stress, enhancing colonic tight junctions, and regulating intestinal flora. This colon-accumulating oral drug delivery nanoplatform may provide a novel and precise therapeutic strategy for UC.

Comparison of the antioxidant activities of nonfumigated and sulphur-fumigated Chrysanthemum morifolium cv. Hang-ju induced by oxidative stress.

CONTEXT: The traditional drying method, sun drying, for Chrysanthemum morifolium Ramat. cv. Hang-ju (Compositae) (HJ) is widely replaced by sulphur fumigation (SF), which has an unknown effect on its efficacy. OBJECTIVE: To investigate protective effects of nonfumigated HJ (NHJ) and sulphur-fumigated HJ (SHJ) water extracts against oxidative stress and lipid peroxidation. MATERIALS AND METHODS: Sprague-Dawley rats were administered high-fat diet to induce hyperlipidaemia and randomly divided into eight groups (n = 6): control, fenofibrate, NHJ and SHJ extracts (1, 2 or 4 g crude drugs/kg/d; intragastric administration for 8 weeks). Serum total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), superoxide dismutase (SOD) and malondialdehyde (MDA) levels were detected. Human umbilical vein endothelial cells (HUVECs) were treated with NHJ and SHJ extracts (50, 100 or 200 µg/mL) for 24 h, followed by oxidized low-density lipoprotein (ox-LDL, 20 µg/mL) for 2 h in vitro. Cellular reactive oxygen species (ROS), SOD and MDA levels and apoptosis were evaluated. RESULTS: NHJ was more effective than SHJ in decreasing serum TG, TC, LDL-C, LDL/HDL and MDA while increasing serum HDL-C and SOD levels at high doses. SHJ (IC50=19.9 mg/mL) suppressed HUVEC growth stronger than NHJ (IC50=186.7 mg/mL). At 200 µg/mL, NHJ was more effective than SHJ in downregulating ROS and MDA levels, reducing HUVECs apoptosis rate and elevating SOD activity in ox-LDL-treated HUVECs. CONCLUSIONS: SF causes oxidative damage and attenuates antioxidative activity in ox-LDL-treated HUVECs, which promotes lipid peroxidation. SF is not recommended for processing HJ.

Anti-diarrhea effects and identification of Musca domestica larvae low molecular weight peptides (LMWP).

Musca domestica larvae have been used clinically to cure children malnutritional stagnation and low molecular weight peptides (LMWP) of Musca domestica larvae showed more useful bioactivities. But there is no report on anti-diarrhea effects and identification of the LWMP. The purposes of this study were clarifying the anti-diarrhea effects by regulating intestinal microecology and identification of LMWP. In anti-diarrhea test, diarrhea mice were administered LMWP by oral gavage. Then rectal stool indicator bacteria were counted also the identification of rectal stool bacteria were determined by PCR-DGGE. In LMWP identification test, GFC and RP-HPLC were used to separate the peptide. Then the single polypeptide was tested by MALDI TOF and N-terminal sequence analysis. The results of anti-diarrhea showed that LMWP was effective in the inhibition diarrhea in mice. And microbial diversity indices showed that LMWP treatment group exhibited a higher number of bands. The identification test showed that LMWP had four main components (10-30KD, S1, S2, S3), and there were 5, 7 and 4 peaks in S1, S2 and S3, respectively. The the molecular weight of S2-5, S3-2 and S3-3 was 877.053D, 877.0631D and 1069.4391D, respectively. And S3-3 was determined as Chain A, Carboxypeptidase G2. So the hypothesis that intestinal microbiological regulation might be one of the potential anti-diarrhea mechanisms of Musca domestica larvae LMWP which had four main components and one of the single polypeptide was identified could be drawn.

Musca domestica Cecropin (Mdc) Alleviates Salmonella typhimurium-Induced Colonic Mucosal Barrier Impairment: Associating With Inflammatory and Oxidative Stress Response, Tight Junction as Well as Intestinal Flora.

Salmonella typhimurium, a Gram-negative food-borne pathogen, induces impairment in intestinal mucosal barrier function frequently. The injury is related to many factors such as inflammation, oxidative stress, tight junctions and flora changes in the host intestine. Musca domestica cecropin (Mdc), a novel antimicrobial peptide containing 40 amino acids, has potential antibacterial, anti-inflammatory, and immunological functions. It remains unclear exactly whether and how Mdc reduces colonic mucosal barrier damage caused by S. typhimurium. Twenty four 6-week-old male mice were divided into four groups: normal group, control group (S. typhimurium-challenged), Mdc group, and ceftriaxone sodium group (Cs group). HE staining and transmission electron microscopy (TEM) were performed to observe the morphology of the colon tissues. Bacterial load of S. typhimurium in colon, liver and spleen were determined by bacterial plate counting. Inflammatory factors were detected by enzyme linked immunosorbent assay (ELISA). Oxidative stress levels in the colon tissues were also analyzed. Immunofluorescence analysis, RT-PCR, and Western blot were carried out to examine the levels of tight junction and inflammatory proteins. The intestinal microbiota composition was assessed via 16s rDNA sequencing. We successfully built and evaluated an S. typhimurium-infection model in mice. Morphology and microcosmic change of the colon tissues confirmed the protective qualities of Mdc. Mdc could inhibit colonic inflammation and oxidative stress. Tight junctions were improved significantly after Mdc administration. Interestingly, Mdc ameliorated intestinal flora imbalance, which may be related to the improvement of tight junction. Our results shed a new light on protective effects and mechanism of the antimicrobial peptide Mdc on colonic mucosal barrier damage caused by S. typhimurium infection. Mdc is expected to be an important candidate for S. typhimurium infection treatment.

Prodigiosin isolated from Serratia marcescens in the Periplaneta americana gut and its apoptosis­inducing activity in HeLa cells.

Serratia marcescens are considered to be abundant and optimal resources for obtaining prodigiosin, which can be isolated from soil, water, plants and air but rarely from insects. In the present study, a strain of Serratia marcescens named WA12­1­18 was isolated from the gut of Periplaneta americana, which was capable of producing high levels of pigment reaching 2.77 g/l via solid fermentation and was identified as prodigiosin by ultraviolet, high performance liquid chromatography (LC), Fourier­transform infrared spectroscopy, LC­mass spectroscopy and nuclear magnetic resonance. The apoptotic tumor cells treated with prodigiosin were examined by 4',6­diamidino­2­phenylindole (DAPI) staining assays and transmission electron microscopy. Flow cytometry (FCM) was utilized to measure the apoptotic rate with Annexin V staining and the expression levels of proteins involved in apoptosis, including B­cell lymphoma 2 (Bcl­2), Bcl­2­associated X (Bax) and caspase­3 were determined by western blot analysis and reverse transcription­quantitative polymerase chain reaction (RT­qPCR). The experimental results revealed that prodigiosin could inhibit the proliferation of HeLa cells and the half­maximal inhibitory concentration values of prodigiosin in HeLa were 2.1, 1.2 and 0.5 µg/ml over 24, 48 and 72 h, respectively. Furthermore, DAPI staining assays and transmission electron microscopy clearly demonstrated that prodigiosin could induce HeLa cell apoptosis. FCM results revealed that the cell apoptotic rates were 19.7±1.4, 23.7±2.4 and 26.2±2.3% following the treatment with 0.5, 1.0 and 2.0 µg/ml prodigiosin for 48 h, respectively. Western blot analysis and RT­qPCR revealed that prodigiosin could activate apoptosis­associated molecules including Bcl­2, Bax and caspase­3. Therefore, the results of the present study demonstrated that the prodigiosin could induce apoptosis in HeLa cells, which may be associated with the upregulation of Bax and caspase­3, the concomitant downregulation of Bcl­2 levels and also triggering the extrinsic apoptotic signaling pathway.

Cardiovascular and respiratory safety evaluation of Musca Domestica larvae low molecular weight peptide in beagle dogs.

Background: Many studies have demonstrated that the water extracts and low-molecular-weight peptide (LMWP) of the Musca domestica larvae contain significant biological activity. However, the cardiovascular and respiratory safety evaluations of LMWP are yet to be sufficiently investigated. Aim: This study focused on the cardiovascular and respiratory safety evaluations of the M. domestica larvae LMWP in beagle dogs. Methods: Direct cardiovascular and respiratory effects of three different doses of the M. domestica larvae LMWP were investigated following only once oral administration in conscious telemetered dogs, whereby ECG, arterial pressure, and respiratory data were collected using the Data Science International telemetric system. Results: The PR, QT, and QTcf intervals were significantly shortened in the medium-dose LMWP treatment group at 3 h after drug administration. Furthermore, no significant differences were observed in any of the corresponding indexes of other treatment groups at different time points compared to those of the control group. P wave, ST segment, R wave, systolic pressure, diastolic pressure, and mean pressure were significantly different, although these differences had no significant dose-effect relationship. Respiratory frequency significantly increased in the medium-dose LMWP treatment group at 8 h after drug administration compared to that of the control group. Respiratory rate and tidal volume showed no significant differences at varying time points among all LMWP treatment groups. Conclusions: No toxicological effects related to cardiovascular and respiratory safety in beagle dogs were observed at any dose level of the M. domestica larvae LMWP.

In vivo and in vitro anti-inflammatory effects of Sophora flavescens residues.

ETHNOPHARMACOLOGICAL RELEVANCE: The dried roots of Sophora flavescens Ait. (Leguminosae) is traditionally used as antipyretic medicine to reduce inflammation. It is well known that alkaloids and flavonoids are the main constituents of S. flavescens. However, the clinical researches and applications of S. flavescens is mainly based on its water-extracted alkaloids, its flavonoids may still remain in residues and have been underused. With development and manufacturing of S. flavescens in recent years, more herb residues are being produced. Since they are typically treated as waste and dumped openly in landfill sites, which can cause pollution, there is a great need to explore these wastes as recyclable resources and increase their added value. To date, whether other bioactive components would be found in the residues of S. flavescens is still unknown. If the extraction method of these active ingredients was established, the residues of S. flavescens could be turned from the harm to a benefit and make great sense of the comprehensive utilization of S. flavescens resources. This study aimed to establish an extraction method of the residues of S. flavescens and investigate the anti-inflammatory effect of it both in vivo and in vitro. MATERIALS AND METHODS: Dried S. flavescens were decocted with distilled water firstly, then the residues were powdered and extracted with ethyl acetate by using ultrasonic wave. HPLC was utilized to analyze the chemical constituents of the water extracts of S. flavescens (WSF) and the ethyl acetate extracts of residues of S. flavescens (RSF). In vivo, the anti-inflammatory effect of WSF and RSF were evaluated using the xylene-induced auricle edema, acetic acid-induced peritoneal permeability and carrageenan-induced hind paw edema methods. In vitro, the inhibitory activities of WSF and RSF on NO, TNF-α, IL-6 and MCP-1 production of LPS-treated RAW264.7 cells were measured. RESULTS: The major ingredients of RSF were flavonoids, while WSF almost had no flavonoids. In vivo, WSF and RSF (200 mg/kg) could significantly inhibit the edema in the xylene-induced mice auricle edema and carrageenan-induced hind paw edema as well as the peritoneal permeability increased by acetic acid. They can also lower production levels of PGE2 in inflamed paw tissues. In vitro experimental results showed that RSF (25-100 µg/mL) could significantly inhibit the release of pro-inflammatory cytokines NO, TNF-α, IL-6 and MCP-1 on LPS-induced RAW264.7 cells. The in vitro suppress effect of WSF had no dose-response relationship. CONCLUSIONS: The residues of S. flavescens had obvious flavonoids with anti-inflammatory activity. This study provided evidence for the reuse of residues from S. flavescens in the food additive, medicine and cosmetic industries.

Simvastatin Modulates Interaction Between Vascular Smooth Muscle Cell/Macrophage and TNF-α-Activated Endothelial Cell.

Cellular interactions between endothelial cell (EC) and vascular smooth muscle cell (VSMC)/macrophages seem to be greatly changed under inflammatory conditions. Although simvastatin could regulate inflammatory transcription factors in EC and VSMC and also could inhibit leukocyte-endothelium interaction, whether it could modulate VSMC/macrophage functions that are induced by tumor necrosis factor-α (TNF-α)-activated EC remained unclear. The purpose of this study was to investigate the effects of simvastatin on VSMC/macrophage functions, which are induced by TNF-α-activated EC in coculture system in vitro. The results showed that under noncontacting conditions, simvastatin could reduce the proliferation, apoptosis, and TNF-α, IL-6, and vascular endothelial growth factor secretion both in VSMC and macrophage, which is induced by TNF-α-activated EC. And a hypothesis that simvastatin regulates the interactions and the soluble factors between EC and VSMC/macrophages could be drawn. And that might be a potential anti-atherosclerosis mechanism of simvastatin.

Luteolin Promotes Cell Apoptosis by Inducing Autophagy in Hepatocellular Carcinoma.

BACKGROUND/AIMS: Hepatocellular carcinoma (HCC) is the most common primary liver malignancy and is a leading cause of cancer-related death worldwide. Luteolin, a flavonoid from traditional Chinese medicine, shows anti-cancer activity in many cancer cells, including HCC. However, the mechanism underlying the action of luteolin in HCC, especially its role in regulating cell autophagy, remains unclear. In the present study, we investigated the role of luteolin in regulating cell autophagy and the role of autophagy in luteolin-induced apoptosis. METHODS: The 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay (MTT) was used to investigate cell viability. Flow cytometry analysis was used to detect the cell cycle and cell apoptosis. Hoechst 33342 staining was used to detect cell apoptosis. Transmission electron microscopy was used to investigate autophagy. qRT-PCR and western blotting were used to detect apoptosis- and autophagy-related mRNAs and proteins. RESULTS: Luteolin reduced the viability of SMMC-7721 cells in a time and dose-dependent manner, and induced significant G0/G1-phase arrest. In addition, the results of flow cytometry analysis and Hoechst 33342 staining showed that luteolin treatment increased the number of apoptotic cells obviously, and the results of qRT-PCR and western blotting showed that luteolin treatment increased caspase 8 and decreased bcl-2 at the mRNA and protein levels. Furthermore, luteolin increased the number of intracellular autophagosomes, promoted LC3B-I conversion to LC3B-II, and increased Beclin 1 expression. Finally, co-treatment with the autophagy inhibitor chloroquine weakened the effects of luteolin on cell apoptosis. CONCLUSION: Luteolin induced apoptosis in human liver cancer SMMC-7721 cells, partially via autophagy. Thus, luteolin could be used as a regulator of autophagy in HCC treatment.

Effect of Musca domestic maggot polypeptide extract on HUVEC dysfunction induced by early-activated macrophages.

CONTEXT: Musca domestica Linn. maggot is a traditional Chinese medicine. In our previous studies, Musca domestica maggot protein-enriched fraction and polypeptide extract (molecular weight <30 kD) were found to reverse endothelial cell dysfunction in atherosclerotic lesions. OBJECTIVE: This study determines whether and how M. domestica maggot polypeptide extract affects the dysfunction of human umbilical vein endothelial cells (HUVEC) induced by macrophages (Mϕ). MATERIALS AND METHODS: HUVEC and early-activated THP-1 Mϕ (incubated with LPS of 1 µg/ml for 2 h) were co-cultured in a Transwell system. The effects of Musca domestica maggot polypeptide extract (with increasing concentrations, i.e., 1.0, 2.5, 5.0, 10.0, 20.0, and 40.0 µg/ml) on the proliferation and migration HUVEC and their secretion of vascular endothelial growth factor (VEGF) were determined by flow cytometry, modified Boyden chamber assay, and enzyme-linked immunosorbent assay (ELISA) after incubation for 24 h. RESULTS: Musca domestica maggot polypeptide extract decreased the proliferation of HUVEC in a concentration-dependent manner, with a 50% effective concentration (EC50) of 22.16 ± 1.48 µg/ml, and effectively inhibited HUVEC migration (EC50 = 35.15 ± 2.03 µg/ml) and VEGF secretion (EC50 = 28.64 ± 1.29 µg/ml). DISCUSSION AND CONCLUSION: Musca domestica maggot polypeptide extract can inhibit the dysfunction of HUVEC induced by early-activated THP-1 Mϕ.

Chloride channel-3 promotes tumor metastasis by regulating membrane ruffling and is associated with poor survival.

The chloride channel-3 (ClC-3) protein is known to be a component of Cl- channels involved in cell volume regulation or acidification of intracellular vesicles. Here, we report that ClC-3 was highly expressed in the cytoplasm of metastatic carcinomatous cells and accelerated cell migration in vitro and tumor metastasis in vivo. High-grade expression of cytoplasmic ClC-3 predicted poor survival in cancer patients. We found that independent of its volume-activated Cl- channel properties, ClC-3 was able to promote cell membrane ruffling, required for tumor metastasis. ClC-3 mediated membrane ruffling by regulating keratin 18 phosphorylation to control ß1 Integrin recycling. Therefore, cytoplasmic ClC-3 plays an active and key role in tumor metastasis and may be a valuable prognostic biomarker and a therapeutic target to prevent tumor spread.

Cecropin suppresses human hepatocellular carcinoma BEL- 7402 cell growth and survival in vivo without side-toxicity.

Conventional chemotherapy against hepatocellular carcinoma typically causes various side effects. Our previous study showed that cecropin of Musca domestica can induce apoptosis in human hepatocellular carcinoma BEL-7402 cells in vitro. However, whether cecropin inhibits BEL-7402 cell in vivo and the question of possible side effects remained undentified. The present study confirmed tumor-inhibitory effects of cecropin in vivo, and furthermore strongly suggested that cecropin cytotoxicity in BEL-7402 cells in vivo may be mainly derived from its pro-apoptotic action. Specifically, we found that cecropin exerted no obvious side effects in tumor-bearing mice as it had no significant hematoxicity as well as visceral toxicity. Therefore, cecropin may be a potential candidate for further investigation as an antitumor agent against hepatocellular carcinoma.

Antihepatitis B virus activity of a protein-enriched fraction from housefly (Musca domestica) in a stable HBV-producing cell line.

Hepatitis B virus (HBV) infection remains a major public health problem. Although several vaccines and therapeutic strategies are currently being implemented to combat HBV virus, effective antiviral therapy against HBV infection has not been fully developed. Alternative strategies and new drugs to combat this disease are urged. Insects and insect derivatives are a large and unexploited source of potentially useful compounds for modern medicine. In the present study, we investigated the first anti-HBV activity of a protein-enriched fraction (PE) from the larvae of the housefly (Musca domestica) in a stable HBV-producing cell line. HBsAg and HBeAg in the culture medium were measured by enzyme-linked immunosorbent assay. HBV-DNA was quantified by fluorescent quantification PCR. HBV core protein was assayed by immunofluorescent staining. Results indicate PE treatment inhibited both HBsAg, HBeAg secretion, and HBV-DNA replication. Furthermore, PE could also suppress HBV core protein expression. PE could be a potential candidate for the development of a novel and effective drug for the treatment of HBV infection.

Construction and characterization of a thermostable whole-cell chitinolytic enzyme using yeast surface display.

To develop a novel yeast whole-cell biocatalyst by yeast surface display technology that can hydrolyze chitin, the chitinaseC gene from Serratia marcescens AS1.1652 strain was cloned and subcloned into the yeast surface display plasmid pYD1, and the recombinant plasmid pYD1/SmchiC was electroporated into Saccharomyces cerevisiae EBY100 cell. Aga2p-SmChiC fusion protein was expressed and anchored on the yeast cell surface by induction with galactose, which was verified by indirect immunofluorescence and Western blotting. The chitinolytic activity of the yeast whole-cell biocatalyst or partially purified enzyme was detected by agar plate clear zone test, SDS-PAGE zymography and dinitrosalicylic acid method. The results showed that the chitinaseC gene from S. marcescens AS1.1652 strain was successfully cloned and expressed on the yeast cell surface, Aga2p-SmChiC fusion protein with molecular weight (67 kDa) was determined. Tests on the effect of temperature and pH on enzyme activity and stability revealed that the yeast whole-cell biocatalyst and partially purified enzyme possessed both thermal stability and activity, and even maintained some activity under acidic and weakly alkaline conditions. The optimum reaction temperature and pH value were set at 52 °C and 5.0, respectively. Yeast surface display technology succeeded in preparing a yeast whole-cell biocatalyst with chitinolytic activity, and the utilization of chitin could benefit from this process of enzyme preparation.

Construction of a novel liver-targeting fusion interferon by incorporation of a Plasmodium region I-plus peptide.

Interferon alpha (IFN α) exerts a multiplicity of biological actions including antiviral, immunomodulatory, and antiproliferative effects. Administration of IFN α is the current treatment for chronic hepatitis B; however, therapy outcome has not been completely satisfactory. The systemic effects of IFN α may account for its low in vivo biological activity and multiple adverse events. The purpose of this study was to design a novel liver-targeting fusion interferon (IFN-CSP) by fusing IFN α2b with a Plasmodium region I-plus peptide, thus targeting the drug specifically to the liver. The DNA sequence encoding IFN-CSP was constructed using improved splicing by overlapping extension-PCR method, and then cloned into the pET-21b vector for protein expression in E. coli BL21 (DE3). The recombinant protein was expressed as a His-tagged protein and purified using a combination of Ni affinity and HiTrap affinity chromatography at a purity of over 95%. The final yield of biologically active IFN-CSP was up to 270 mg/L culture. The purified recombinant protein showed anti-HBV activity and liver-targeting potentiality in vitro. These data suggests that the novel fusion interferon IFN-CSP may be an excellent candidate as a liver-targeting anti-HBV agent.