Green Extraction of Polyphenols from Elaeagnus angustifolia L. Using Natural Deep Eutectic Solvents and Evaluation of Bioactivity.

In the study, natural deep eutectic solvents (NADESs) were used as alternatives to traditional chemical solvents for the extraction of polyphenols from Elaeagnus angustifolia L. Nine NADESs were tested for the first time and compared with ethanol and water (traditional solvents) regarding the extraction of phenolic compounds from E. angustifolia L. These solvents were particularly effective at extracting polyphenols, whose low water solubility usually requires high amounts of organic solvents. The solvent based on choline chloride and malonic acid provided optimal results and was selected for further optimization. The effects of material-to-liquid ratio, ultrasound time, and ultrasound temperature on the extraction efficiency were studied through single-factor experiments. These parameters were optimized by Box-Behnken design using response surface methodology. The optimal conditions identified were 49.86 g/mL of material-to-liquid ratio, 31.10 min of ultrasound time, and 62.35 °C of ultrasound temperature, resulting in a high yield of 140.30 ± 0.19 mg/g. The results indicated that the NADES extraction technique provided a higher yield than the conventional extraction process. The antioxidant activity of the extract of polyphenols from E. angustifolia L. was determined, and UPLC-IMS-QTOF-MS was used to analyze the phenolic compounds in it. The results revealed that the scavenging ability of 1,1-diphenyl-2-picryl-hydrazil and 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonate) extracted by NADES was higher than that of polyphenols extracted by water and ethanol. Furthermore, a total of 24 phenolic compounds were identified in the extract. To the best of our knowledge, this is the first study in which a green and efficient NADES extraction method has been used to extract bioactive polyphenols from E. angustifolia L., which could provide potential value in pharmaceuticals, cosmetics, and food additives.

Effect of Pressure and Nonpressure Dressings on Postoperative Complications in Patients With Mixed Hemorrhoids: A Single-blind Controlled Study.

PURPOSE: To compare the clinical effects of nonpressure and pressure dressings on the postoperative complications of modified Milligan-Morgan hemorrhoidectomy. DESIGN: Randomized controlled trial. METHODS: A total of 186 patients with grade II to III mixed hemorrhoids who had been excluded from cardiovascular and cerebrovascular diseases and anorectal surgery were included and randomly assigned to the nonpressure dressings group and the pressure dressings group by random number table. The incidence of acute urinary retention and medical adhesive-related skin injury, pain, hemostatic effect, anal distension, anal edema, use of analgesics, length of hospital stay, and hospitalization costs were compared between the two groups. The Consolidated Standards of Reporting Trials checklist for randomized controlled trials was used in this study. FINDINGS: The incidence of acute urinary retention in both men and women was significantly lower in the nonpressure dressing group (relative risk [RR] = 0.20, 95% confidence interval [CI] [0.13, 0.37], P = .002); (RR = 0.47, 95% CI [0.22, 0.76], P = .015). The postoperative pain at 6 hours/18 hours/25 hours was significantly lower in the nonpressure dressing group (P < .001, P = .004 < 0.05, P = .009). The anal distension at 6 hours and the number of patients who used analgesics were significantly lower in the nonpressure dressing group (P < .001). The incidence of medical adhesive-related skin injuries was significantly lower in the nonpressure dressing group (RR = 0.061, 95% CI [0.020, 0.189], P < .001). No primary bleeding was observed in both groups. However, there were no significant differences between both groups in terms of anal edema scores, length of stay, or hospitalization expenses. No adverse events were reported in either group during the study period. CONCLUSIONS: Nonpressure dressings can effectively reduce the incidence of acute urinary retention and medical adhesion-related skin injury after surgery for grade III to IV mixed hemorrhoids. They can also safely relieve pain and distension.

Genome mining discovery of hydrogen production pathway of Klebsiella sp. WL1316 fermenting cotton stalk hydrolysate.

Genome sequencing was used to identify key genes for the generation of hydrogen gas through cotton stalk hydrolysate fermentation by Klebsiella sp. WL1316. Genome annotation indicated that the genome size was 5.2 Mb with GC content 57.6%. Xylose was metabolized in the pentose phosphate pathway via the conversion of xylose to xylulose in Klebsiella sp. WL1316. This strain contained diverse formate-hydrogen lyases and hydrogenases with gene numbers higher than closely related species. A metabolic network involving glucose, xylose utilisation, and fermentative hydrogen production was reconstructed. Metabolic analysis of key node metabolites showed that glucose and xylose metabolism influenced biomass synthesis and biohydrogen production. Formic acid accumulated during fermentation at 24-48 h but decreased sharply after 48 h, illustrating the splitting of formic acid to hydrogen gas during early-to-mid fermentation. The Kreb's cycle was the main competitive metabolic branch of biohydrogen synthesis at 24 h of fermentation. Lactic and acetic acid fermentation and late ethanol accumulation competed the carbon skeleton of biohydrogen synthesis after 72 h of fermentation, indicating that these competitive pathways are regulated in middle-to-late fermentation (48-96 h). This study is the first to elucidate the metabolic mechanisms of mixed sugar utilisation and biohydrogen synthesis based on genomic information.

miRNA-17-92 protects endothelial cells from erastin-induced ferroptosis through targeting the A20-ACSL4 axis.

Endothelial cell death is linked to vascular diseases such as atherosclerosis and tissue ischemia. miRNA-17-92 (miR-17-92) is a multiple functional oncogenic miRNA cluster which plays vital roles in tumor angiogenesis and tissue development. However, its role in regulation of endothelial cell ferroptosis remains unclear. In this study, we revealed that miR-17-92 protects endothelial HUVEC cells from erastin-induced ferroptosis. miR-17-92 overexpression significantly reduced erastin-induced growth inhibition and ROS generation of HUVEC cells. Furthermore, Zinc lipoprotein A20, a validated target of miR-17-92, was identified as a novel regulator of endothelial cell ferroptosis. Lentivirus mediated A20 overexpression increased ROS generation and enhanced erastin-induced ferroptosis, whereas A20 knockdown inhibited erastin-induced ferroptosis. Mechanistic studies revealed that erastin-induced ferroptosis is associated with GPX4 downregulation and ACSL4 upregulation. miR-17-92 overexpression or A20 inhibition increased the ACSL4 expression in HUVEC cells. A20 was identified to directly with and regulate ACSL4 expression by immunoprecipitation. It suggests that the A20-ACSL4 axis plays important roles in erastin-induced endothelial ferroptosis. In conclusion, this study revealed a novel mechanism through which miR-17-92 protects endothelial cells from erastin-induced ferroptosis by targeting the A20-ACSL4 axis.

miR-17-92 promotes leukemogenesis in chronic myeloid leukemia via targeting A20 and activation of NF-κB signaling.

miR-17-92 cluster are overexpressed in hematological malignancies including chronic myeloid leukemia (CML). However, their roles and mechanisms that regulate BCR-ABL induced leukemogenesis remain unclear. In this study, we demonstrated that genomic depletion of miR-17-92 inhibited the BCR-ABL induced leukemogenesis by using a mouse model of transplantation of BCR-ABL transduced hematopoietic stem cells. Furthermore, we identified that miR-19b targeted A20 (TNFAIP3). A20 overexpression results in inactivation of NF-κB activity including decrease of phosphorylation of P65 and IκBα, leads to induce apoptosis and inhibit proliferation and cycle in CML CD34 + cells. Thus we proved that miR-17-92 is a critical contributor to CML leukemogenesis via targeting A20 and activation of NF-κB signaling. These findings indicate that miR-17-92 will be important resources for developing novel treatment strategies of CML and better understanding long-term disease control.

SUMO-Specific Cysteine Protease 1 Promotes Epithelial Mesenchymal Transition of Prostate Cancer Cells via Regulating SMAD4 deSUMOylation.

In advanced prostate cancer, small ubiquitin-like modifier (SUMO)-specific cysteine protease 1 (SENP1) is up-regulated. However, the role of SENP1 in regulating deSUMOylation of TGF-ß/SMADs signaling is unknown. In this study, we developed a lentiviral vector, PLKO.1-shSENP1, to silence SENP1 in prostate cancer cells with high metastatic characteristics (PC3M). Likewise, we also created an adenovirus vector, Ad5/F11p-SENP1 to over-express SENP1 in prostate cancer cells with low metastatic potential (LNCaP). We showed that silencing of SENP1 promoted cellular apoptosis, and inhibited proliferation and migration of PC3M cells. Moreover, SENP1 silencing increased the SMAD4 expression at protein level, up-regulated E-cadherin and down-regulated Vimentin expression, indicating the inhibition of epithelial mesenchymal transition (EMT). Furthermore, SMAD4 interference abolished SENP1-mediated up-regulation of E-cadherin, suggesting that SENP1 regulated E-cadherin expression via SMAD4. SENP1 over-expression in LNCaP cells reduced SMAD4 protein, and promoted EMT via decreasing E-cadherin and increasing Vimentin. Moreover, down-regulation of SMAD4 and E-cadherin were blocked, after transfection with two SUMOylation sites mutated SMAD4, suggesting that SENP1 might reduce SMAD4 levels to regulate E-cadherin expression via deSUMOylation of SMAD4. In conclusion, SENP1 deSUMOylated SMAD4 to promote EMT via up-regulating E-cadherin in prostate cancer cells. Therefore, SENP1 is a potential target for treatment of advanced prostate cancer.

Effect of oral hepatocyte growth factor gene mediated by attenuated salmonella on 2-, 4-, 6-trinitro-benzene-sulfonic-acid-induced ulcerative colitis in rat.

BACKGROUND AND AIM: In order to explore a new therapeutic method, we investigated the effects of exogenously expressed hepatocyte growth factor mediated by attenuated salmonella (TPH) on rats with ulcerative colitis (UC) induced by 2-, 4-, 6-trinitro-benzene-sulfonic acid. METHODS: The UC rats were treated with TPH, attenuated salmonella with a eukaryotic expression vector (TP) or sodium bicarbonate (model control [MC]) every other day. Cluster of differentiation (CD)4(+) and CD8(+) T cells and immunoglobulins in the blood were analyzed by flow cytometry. The HGF expression was determined by immunohistochemistry. A macroscopic-scale observation of the colon and a histological assessment were also carried out. RESULTS: The CD4(+) T counts and the CD4(+) /CD8(+) ratio in the TPH group were significantly lower than that in the MC group. The immunoglobulin M and immunoglobulin G(1) levels in the TPH group were significantly lower than that in the MC group and TP group. After treatment with TPH, the symptoms of the ulcerative rats were significantly alleviated. The colonic lesion grades in the TPH group were lower than that in the TP group and MC group. Significant improvement occurred after the TPH treatment, as evidenced by alleviated mucosal inflammation. At 7 days post-treatment, the HGF expression in the colonic tissues that were treated with TPH was stronger than that in the samples treated with TP. CONCLUSIONS: TPH inhibits the proliferation of T lymphocytes and the antibody production of B lymphocytes. Furthermore, it ameliorates mucosal inflammation and promotes the regeneration of mucosa and the healing of the colonic ulceration.

Oxidative stress: a possible mechanism for lead-induced apoptosis and nephrotoxicity.

Lead-induced nephrotoxicity is a human health hazard problem. In this study, Human mesangial cells (HMCs) were treated with different concentration of lead acetate (5, 10, 20 µmol/l) in order to investigate the oxidative stress and apoptotic changes. It was revealed that lead acetate could induce a progressive loss in HMCs viability together with a significant increase in the number of apoptotic cells using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium (MTT) assay and flow cytometry, respectively. The apoptotic morphological changes induced by lead exposure in HMCs were demonstrated by PI-Hochest33342 staining. A DNA laddering pattern in lead-treated cells was shown, which could indicate nuclear fragmentation. In addition, lead acetate significantly increased the levels of malondialehyde (MDA) content and lactate dehydrogenase (LDH) activity. Therefore, it might be concluded that lead could promote HMCs' oxidative stress and apoptosis, which may be the chief mechanisms of lead-induced nephrotoxicity.

[The toxic effects of lead acetate on the apoptosis and the ultrastructure in human renal tubular epithelial cells (HK-2)].

OBJECTIVE: To explore the toxic effects of lead acetate on the apoptosis and ultrastructure of human renal tubular epithelial cells (HK-2). METHODS: After HK-2 cells were exposed to 5, 10 and 20 µmol/L lead acetate for 24 h, the morphological changes of HK-2 cells were observed by Hochest 33342-PI staining, and the ultrastructure changes of HK-2 cells were examined under a electron microscope, LDH activity and MDA content in supernatant of HK-2 cellular culture were detected by spectrophotometer, DNA damage of HK-2 was determined by DNA ladder and the apoptotic rates of HK-2 cells were measured by flow cytometry. RESULTS: The morphological changes of apoptotic HK-2 cells in exposure group were observed by Hochest 33342-PI staining. The cytoplasm vacuoles, karyopycnosis, nuclear membrane vague and apoptotic bodies in HK-2 cells of exposure group were found under electron microscopy. LDH activity and MDA contents in exposure group increased significantly, as compared to control group (P < 0.01). The results of DNA Ladder showed that DNA damage of HK-2 cells in exposure group appeared. The apoptotic rates of HK-2 cells exposed to 5, 10, 20 µmol/L lead acetate were 14.16% ± 2.94%, 19.45% ± 2.73%, 25.01% ± 3.97%, respectively, which were significantly higher than that (5.81% ± 2.18%) in control group (P < 0.05). CONCLUSION: Lead acetate could remarkably induce the apoptosis of HK-2 cells and affect the kidney.

[The effects of lead exposure on primary cultured hippocampus neurons apoptosis and the expressions of Bcl-2, Bax, p53 and NOS1].

OBJECTIVE: To observe effects of plumbum on the apoptosis of primary cultured hippocampus neurons. METHODS: After primary cultured hippocampus neurons of rats treated with different concentration of Pb (C2H3O2) (10, 50, 100 and 200 microg/ml), to observe the morphological changes of the apoptosis of hippocampus cells by Giemsa dyeing, to detect the rate of apoptosis by flow cytometry, to observe the expression of Bcl-2, Bax and p53 by immunocytochemical stain and to analyze quantitatively the average fluorescence intensity of free Ca2+ in kytoplasm of each concentration group by laser scanning confocal microscope (LSCM). RESULTS: The results of Giemsa dyeing showed typically morphological changes of apoptosis after hippocampus neurons treated with certain concentration of Pb(C2H3O2), and had dose-dependent; The results of flow cytometry showed,after cells treated respectively in 50, 100, 200 microg/ml for 24h, that the rate of apoptosis increased and compared with the control group the difference of were all statistical significance (P < 0.01); The results of immunocytochemical stain showed that, with dose of Pb increasing, the expression of Bcl-2 protein decreased, the intensity of dyeing increasingly degraded and the expression of Bax and p53 was up-regulation, and compared with the control group, IOD value of cells had statistical significance (P < 0.01); The results of LSCM showed that the concentration of Ca2+ in kytoplasm was also increased with the dose of Pb (C2H3O2) increasing, and the average fluorescence intensity in groups of three concentration of Pb(C2H3O2) was all significantly higher than in the control group (P < 0.01) and had dose-response relation. CONCLUSION: Plumbum might lead to different degrees of the apoptosis of hippocampus neurons. It is supposed that Pb in certain concentration range might affect the normal function of hippocampus neurons in learning and memory, and so on.

Newly synthesized podophyllotoxin derivative, LJ12, induces apoptosis and mitotic catastrophe in non-small cell lung cancer cells in vitro.

Deoxypodophyllotoxin (DPT), an active compound isolated from a number of herbs and used in traditional medicine, has been reported to exhibit promising anti­tumor activity. A newly synthesized derivative, N-(1-oxyl­4'-demethyl-4-deoxyp odophyllic)-L­methine-4'-piperazine carbamate (LJ12) may have improved antitumor activity and fewer side effects. The present study assessed the effect of LJ12 on cell viability, apoptosis, cell cycle distribution and mitotic catastrophe in A549 human lung cancer cells in vitro. The molecular mechanisms underlying the antitumor activity of LJ12 were also examined. The results demonstrated that LJ12 reduced A549 cell viability in a time­ and dose­dependent manner, with a lower half maximal inhibitory concentration of ~0.1 µM, compared with another known DPT derivative, etoposide (10 µM). Flow cytometric analysis showed that LJ12 induced tumor cell arrest at the G2/M phase of the cell cycle. The present study also observed an expected concomitant decrease in the numbers of cells cells in the G0/G1 and S phases. LJ12 was found to upregulate the protein expression levels of Cdc2 and Cyclin B1. Furthermore, LJ12 induced tumor cell apoptosis and the protein expression of B cell lymphoma­2­associated X protein, caspase­3 and p53. The present study also observed the formation of giant, multinucleated cells, indicating that LJ12 induced mitotic catastrophe in the tumor cells. These results indicated that LJ12 has anti­non­small cell lung cancer activity in vitro. Further investigations aim to develop LJ12 as a therapeutic agent for the treatment of lung cancer.

Hepatocyte growth factor protects human mesangial cells against apoptosis induced by lead damage.

Lead is a kind of nephrotoxic metal which frequently threats human health. Hepatocyte growth factor (HGF) is a multifunctional growth factor that protects cell apoptosis. In this study, human mesangial cells (HMCs) were treated with a single HGF dose of 20 and 40 µl/ml in order to investigate the effect of HGF on proliferation and apoptosis ability of HMCs induced by lead acetate. In HGF-treated group, HMCs were incubated with HGF (20, 40 µl/ml) half an hour prior to lead inducing. After lead-induced damage 48 h, the proliferation of HMCs was measured by MTT assay, and the apoptosis was assessed by flow cytometry. RT-PCR was used to detect the expression of P53, Bcl-2, Bax, and caspase-3 mRNA. The expression of Bax protein was measured by Western blot analysis. The results showed that HGF inhibits proliferation of HMCs induced lead acetate in a dose-dependent manner (P < 0.05). HGF significantly promoted the proliferation of HMCs, and flow cytometry revealed that HGF can inhibit apoptosis of HMCs. RT-PCR and Western blot showed that P53, Bax, and caspase-3 expression decreased, while Bcl-2 expression increased. HGF may afford a protection to HMCs against lead-induced damage.

[Study on bone marrow mesenchymal stem cells transfected with adenovirus hepatocyte growth factor gene promoting wounds repair in diabetic rats].

OBJECTIVE: To explore the effects of bone marrow mesenchymal stem cells (BMSCs) transfected with adenovirus hepatocyte growth factor (Ad-HGF) on wound repair in diabetic rats. MRTHODS: BMSCs from male Wistar rats were isolated by density gradient centrifugation, cultured, and transfected with Ad-HGF. The multiplicity of infection was 100. Diabetic models were established in 20 female Wistar rats by diets in high fat and sugar plus intraperitoneal injection of streptozotocin (30 mg/kg). Then 2 full-thickness skin wounds (approximately 1.5 cm in diameter) were made on the dorsum. The rats were randomly divided into 4 groups (n = 5 rats). After wounding, the 0.3 mL suspensions of BMSCs (group A), Ad-HGF (group B), BMSCs transfected with Ad-HGF (group C), and PBS (group D) were injected directly into the dermal of wounds. The transverse diameter and longitudinal diameter of wound were measured at 21 days after treatment. At 7 days and 28 days after treatment, HE staining was performed to evaluate wound healing. The contents of hydroxyproline and advanced glycosylation end products (AGEs) in the wounds were measured by enzyme linked immunosorbent assay and fluorospectrophotometer, respectively, at 3, 7, 14, and 28 days after treatment. RESULTS: At 21 days after treatment, the wounds almost healed in group C, and the transverse diameter and longitudinal diameter were 0 and (0.110 +/- 0.024) cm, respectively. But the wounds healed partially in groups A, B, and D, and the transverse diameter and longitudinal diameter were (0.470 +/- 0.051) cm and (0.590 +/- 0.041) cm, (0.390 +/- 0.042) cm and (0.480 +/- 0.032) cm, and (0.700 +/- 0.068) cm and (0.820 +/- 0.068) cm, respectively. There were significant differences in wound healing between group C and groups A, B, and D (P < 0.05). The wound healing time of group C [(20.5 +/- 1.9) days] was significantly shorter (P < 0.05) than those of groups A, B, and D [(28.3 +/- 1.9), (25.9 +/- 2.3), and (36.6 +/- 5.1) days]. At 7 days, the HE staining showed that evident epidermis transportation, collagen formation, and leukocytes infiltration were observed in group C. At 28 days, the HE staining showed that the epidermis in group C was significantly thinner and more regular than those in other groups, and the decreased collagen and many small vessels were observed in group C. The content of hydroxyproline in group C was higher than those in groups A, B, and D at 7 days and 14 days (P < 0.05). The contents of AGEs in group C was lower than those in groups A, B, and D at 14 days and 28 days (P < 0.05). CONCLUSION Transplantation of BMSCs transfected with Ad-HGF can accelerate the wounds repair in diabetic rats.