Phenolic constituents from the branches of Viburnum chinshanense as potential α-amylase and α-glucosidase inhibitory agents.

This paper reports the isolation of two undescribed phenolic glycosides (1 and 2), together with seven known compounds (3-9) from the branches of Viburnum chinshanense. The structures of undescribed compounds were elucidated by comprehensive spectroscopic methods (1D NMR, 2D NMR, and HRESIMS). The sugar units of compounds 1 and 2 were identified by acid hydrolysis and HPLC analysis of the chiral derivatives of the monosaccharides. Furthermore, the α­amylase and α-glucosidase inhibitory activities of all isolates were evaluated and compounds 1, 5, and 8 displayed potential α­amylase and α-glucosidase inhibitory activities. The molecular docking analyses of compounds 1 and 8 with the potent inhibition towards the target enzymes were also performed.

Quality appraisal and descriptive analysis of clinical practice guidelines for self-managed non-pharmacological interventions of cardiovascular diseases: a systematic review.

BACKGROUND: Cardiovascular diseases (CVDs) are the leading cause of death around the world. Most CVDs-related death can be prevented by the optimal management of risk factors such as unhealthy diet and physical inactivity. Clinical practice guidelines (CPGs) for CVDs, provide some evidence-based recommendations which help healthcare professionals to achieve the best care for patients with CVDs. This systematic review aims to appraise the methodological quality of CPGs systematically and summarize the recommendations of self-managed non-pharmacological interventions for the prevention and management of CVDs provided by the selected guidelines. METHODS: A comprehensive electronic literature search was conducted via six databases (PubMed, Medline, The Cochrane Library, Embase, CINAHL, and Web of Science), seven professional heart association websites, and nine guideline repositories. The Appraisal of Guidelines, Research and Evaluation II (AGREE II) instrument was adopted to critically appraise the methodological quality of the selected guidelines. Content analysis was used to summarise recommended self-managed non-pharmacological interventions for CVDs. RESULTS: Twenty-three CPGs regarding different CVDs were included, in which four guidelines of CVDs, three for coronary heart diseases, seven for heart failure, two for atrial fibrillation, three for stroke, three for peripheral arterial disease, and one for hypertrophic cardiomyopathy. Twenty CPGs were appraised as high quality, and three CPGs as moderate quality. All twenty-three CPGs were recommended for use with or without modification. The domain of "Editorial Independence" had the highest standardized percentage (93.47%), whereas the domain of "Applicability" had the lowest mean domain score of 75.41%. The content analysis findings summarised some common self-managed non-pharmacological interventions, which include healthy diet, physical activity, smoking cessation, alcohol control, and weight management. Healthy diet and physical acidity are the most common and agreed on self-managed interventions for patients with CVDs. There are some inconsistencies identified in the details of recommended interventions, the intervention itself, the grade of recommendation, and the supported level of evidence. CONCLUSION: The majority of the summarized non-pharmacological interventions were strongly recommended with moderate to high-quality levels of evidence. Healthcare professionals and researchers can adopt the results of this review to design self-managed non-pharmacological interventions for patients with CVDs.

Lignan and phenolic glycosides from Viburnum betulifolium fruits and their α­amylase and α-glucosidase inhibitory activities.

The phytochemical investigation on the methanol extract of Viburnum betulifolium fruits resulted in the isolation and identification of two new lignan constituents (1 and 2) and seven known phenolic glycosides (3-9). The structures of new isolates, including their absolute configurations were elucidated by extensive spectroscopic analyses (1H and 13C NMR, HSQC, HMBC, HRESIMS, and ECD) and chemical methods. In the in vitro enzyme assays, compounds 1, 2, 6, and 8 showed potential α­amylase and α-glucosidase inhibitory activities. Among them, compound 1 exhibited stronger inhibitory effects towards α-amylase and α-glucosidase with the IC50 values of 12.68 and 15.17 µM, respectively, than those of the positive control acarbose (IC50, 29.19 and 18.15 µM, respectively). In addition, the molecular docking analyses of compound 1 with strongest inhibition against the target enzymes were also performed.

LC-MS guided isolation of phenolic glycosides from Viburnum luzonicum Rolfe leaves and their α­amylase and α-glucosidase inhibitory activities.

Viburnum luzonicum Rolfe is widely used in China as folk medicine. The bioactivity evaluation indicated that the n-BuOH fraction of V. luzonicum leaves (VLLB) could significantly inhibit α­amylase and α-glucosidase. In order to clarify its active constituents, the phytochemical analysis on VLLB was first performed using HPLC-QTOF-MS/MS, and three new phenolic compounds, viburosides A-C (1-3), along with seven known analogues (4-10) were isolated through preparative HPLC. The undescribed compounds were determined by extensive spectroscopic analyses (1H and 13C NMR, HSQC, HMBC, HRESIMS, and ORD) and enzymatic hydrolysis. In the in vitro enzyme assays, compounds 1-8 showed potent α­amylase and α-glucosidase inhibitory activities. The enzymatic kinetics and molecular docking of the strongest inhibitors 2 and 3 against the corresponding target enzyme were also performed.

Influencing factors of medical device-related pressure ulcers in medical personnel during the COVID-19 pandemic: A systematic review and meta-analysis.

OBJECTIVE: To determine the influencing factors of medical device related pressure injury (MDRPU) in medical staff by meta-analysis. METHODS: A comprehensive literature search was conducted by PubMed, Embase, Cochrane Library, Web of Science, CNKI, VIP, CBM, and WanFang Data (from inception to July 27, 2022). Two researchers independently performed literature screening, quality evaluation and data extraction, and meta-analysis was conducted with RevMan 5.4 and Stata12.0 software. RESULTS: Total of 11215 medical staff were included in 9 articles. Meta analysis showed that gender, occupation, sweating, wearing time, single working time, department of COVID-19, preventive measures, and level 3 PPE were the risk factors for MDRPU in medical staff (P < 0.05). CONCLUSION: The outbreak of COVID-19 led to the occurrence of MDRPU among medical staff, and the influencing factors should be focused on. The medical administrator can further improve and standardize the preventive measures of MDRPU according to the influencing factors. Medical staff should accurately identify high-risk factors in the clinical work process, implement intervention measures, and reduce the incidence of MDRPU.

Effects of exercise interventions on cancer-related fatigue in breast cancer patients: an overview of systematic reviews.

OBJECTIVE: This overview of systematic reviews aims to critically appraise and consolidate evidence from current systematic reviews (SRs)/meta-analyses on the effects of exercise interventions on cancer-related fatigue (CRF) in breast cancer patients. METHODS: SRs/meta-analyses that explored the effects of exercise interventions on CRF in breast cancer patients compared with the routine methods of treatment and care were retrieved from nine databases. The methodological quality of the included SRs was appraised using A MeaSurement Tool to Assess systematic Reviews II (AMSTAR II). The Grading of Recommendations Assessment, Development and Evaluation (GRADE) was used to calculate the grading of outcomes in the included SRs. The exercise type, frequency, duration, and inclusion/absence of supervision were further evaluated with subgroup analyses. The Stata 16.0 software was utilized for data analysis. RESULTS: Twenty-nine reviews were included. The overall methodological quality and level of evidence of the included reviews were unsatisfactory, with only three reviews rated as high methodological quality and no review identified as high-quality evidence. Moderate certainty evidence indicated that exercise could improve fatigue in breast cancer patients (SMD = - 0.40 [95%CI - 0.58, - 0.22]; P = 0.0001). Subgroup analysis based on the types of exercise showed that yoga (SMD = - 0.30 [95%CI - 0.56, - 0.05]; I2 = 28.7%) and aerobic exercise (SMD = - 0.29 [95%CI - 0.56, - 0.02]; I2 = 16%) had a significantly better effect on CRF in breast cancer patients; exercising for over 6 months (SMD = - 0.88 [95%CI - 1.59, - 0.17]; I2 = 42.7%; P = 0.0001), three times per week (SMD = - 0.77 [95%CI - 1.04, - 0.05]; I2 = 0%; P = 0.0001), and for 30 to 60 min per session (SMD = - 0.81 [95%CI - 1.15, - 0.47]; I2 = 42.3%; P = 0.0001) can contribute to a moderate improvement of CRF. Supervised exercise (SMD = - 0.48 [95%CI - 0.77, - 0.18]; I2 = 87%; P = 0.001) was shown to relieve CRF. CONCLUSION: Exercise played a favorable role in alleviating CRF in breast cancer. Yoga was recommended as a promising exercise modality for CRF management in the majority of the included studies. Exercising for at least three times per week with 30 to 60 min per session could be recommended as a suitable dosage for achieving improvement in CRF. Supervised exercise was found to be more effective in alleviating CRF than unsupervised exercise. More rigorously designed clinical studies are needed to specify the exact exercise type, duration, frequency, and intensity to have an optimal effect on CRF in breast cancer patients. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: CRD42020219866.

Self-Managed Non-Pharmacological Interventions for Breast Cancer Survivors: Systematic Quality Appraisal and Content Analysis of Clinical Practice Guidelines.

Background: A growing number of clinical practice guidelines (CPGs) regarding non-pharmacological interventions for breast cancer survivors are available. However, given the limitations in guideline development methodologies and inconsistent recommendations, it remains uncertain how best to design and implement non-pharmacological strategies to tailor interventions for breast cancer survivors with varied health conditions, healthcare needs, and preferences. Aim: To critically appraise and summarise available non-pharmacological interventions for symptom management and health promotion that can be self-managed by breast cancer survivors based on the recommendations of the CPGs. Methods: CPGs, which were published between January 2016 and September 2021 and described non-pharmacological interventions for breast cancer survivors, were systematically searched in six electronic databases, nine relevant guideline databases, and five cancer care society websites. The quality of the included CPGs was assessed by four evaluators using The Appraisal of Guidelines for Research and Evaluation, second edition tool. Content analysis was conducted to synthesise the characteristics of the non-pharmacological interventions recommended by the included CPGs, such as the intervention's form, duration and frequency, level of evidence, grade of recommendation, and source of evidence. Results: A total of 14 CPGs were included. Among which, only five were appraised as high quality. The "range and purpose" domain had the highest standardized percentage (84.61%), while the domain of "applicability" had the lowest (51.04%). Five CPGs were rated "recommended", seven were "recommended with modifications", and the other two were rated "not recommended". The content analysis findings summarised some commonly recommended self-managed non-pharmacological interventions in the 14 guidelines, including physical activity/exercise, meditation, hypnosis, yoga, music therapy, stress management, relaxation, massage and acupressure. Physical activity/exercise was the most frequently recommended approach to managing psychological and physical symptoms by the included guidelines. However, significant variations in the level of evidence and grade of recommendation were identified among the included CPGs. Conclusion: Recommendations for the self-managed non-pharmacological interventions were varied and limited among the 14 CPGs, and some were based on medium- and low-quality evidence. More rigorous methods are required to develop high-quality CPGs to guide clinicians in offering high-quality and tailored breast cancer survivorship care.

Clinical practice guidelines for the nutritional risk screening and assessment of cancer patients: a systematic quality appraisal using the AGREE II instrument.

PURPOSE: To evaluate the quality of published clinical practice guidelines (CPGs) regarding the nutritional risk screening and assessment of cancer patients and to identify high-quality CPGs for clinical healthcare professionals. METHODS: Guidelines for the nutritional risk screening and assessment of cancer patients were comprehensively searched in eight electronic databases, including The Lancet, PubMed, Cochrane Library, Excerpta Medica dataBASE (EMBASE), Web of Science, China National Knowledge Infrastructure (CNKI), China Biology Medicine disc (CBMdisc), and Wan Fang Data, through August 2020. Six relevant guideline databases, including the National Comprehensive Cancer Network (NCCN), the National Guideline Clearinghouse (NGC), the Guideline International Network (GIN), the New Zealand Guidelines Group (NZGG), the China Guideline Clearinghouse (CGC), and Medlive, and relevant nutrition society websites, were also searched through August 2020. The methodological quality of the included CPGs was appraised independently by three assessors using the Appraisal of Guidelines for Research and Evaluation, 2nd edition (AGREE II) tool. RESULTS: Seven CPGs were located, and the domain with the highest percentage was "clarity of presentation" (85.44%), while the domain with the lowest percentage was "applicability" (40.26%). From the AGREE II results, two guidelines were rated as "strongly recommended," three were assessed as "recommended with modifications," and two were deemed as "not recommended." CONCLUSION: Considering that the two "strongly recommended" guidelines were developed within the American and European contexts, translation, validation, and cultural adaptation are recommended prior to implementing these guidelines in other countries or healthcare contexts to improve their effectiveness and sensitivity for local cancer patients. TRIAL REGISTRATION: PROSPERO registration of the study protocol: CRD42020177390 (July 5, 2020).

[Effect of Coixenolide on Foxp3+ CD4+ CD25+ Regulatory T Cells in Collagen-induced Arthritis Mice].

OBJECTIVE: To study the effect of coixenolide on Foxp3+ CD4+ CD25+ regulatory T cells (Treg) in collagen induced arthritis (CIA) mice, and to explore its possible mechanism for treating rheumatiol arthritis. METHODS: Five mice were recruited as a normal control group from 25 mice, and the rest 20 were used in CIA modeling. After successful modeling they were randomly divided in the model control group and the coixenolide group, 10 in each group. Coixenolide injection at 25 mL/kg was intraperitoneally injected to mice in the coixenolide group, while normal saline at 25 mL/kg was intraperitoneally injected to mice in the normal control group and the model control group. The injection lasted for 21 days. Scoring for CIA was performed after injection and arthritis index was calculated. The peripheral blood Foxp3+ CD4+ CD25+ Treg ratio was determined by flow cytometry (FCM). RESULTS: Compared with the normal control group, the arthritis index obviously increased in the model control group (P < 0.01). The arthritis index obviously decreased more in the coixenolide group than in the model control group (P < 0.01). Foxp3+ CD4+ CD25+ Treg levels obviously decreased more in the model control group than in the normal control group (P < 0.01 ). Foxp3+ CD4+ CD25+ Treg levels obviously increased more in the coixenolide control group than in the model control group (P < 0.01). CONCLUSION: Coixenolide could up-regulate Foxp3+ CD4+ CD25+ Treg ratios in CIA mice, which might play certain immunoregulation roles in the incidence of CIA.

[Effects of Wenhua Juanbi Recipe on the Expressions of DNA Methyltransferases in Peripheral Blood Mononuclear Cells of Collagen-Inducing Arthritis Rats].

Objective To observe the effect of Wenhua Juanbi Recipe (WJR) on the expres- sions of DNA methyltransferases (DNMTs) in peripheral blood mononuclear cells (PBMCs) of collagen- inducing arthritis (CIA) , and to study its mechanism for treating CIA. Methods Totally 90 Wistar rats were randomly divided into the model group (n =80) and the normal control group (n = 10). Rats of the model group were injected with type II collagen of bovine (BC II) emulsion from the tail to establish CIA model. Successfully modeled 50 CIA rats were randomly divided into five groups, i.e., the model group, the methotrexate (MTX) group, the low dose WJR group, the middle dose WJR group, the high dose WJR group, 10 in each group. Rats in the model group were administered with normal saline by gastrogavage, once per day. Rats in low, middle, and high dose WJR groups were administered with WJR by gas- trogavage at the daily dose of 22. 9, 45. 8, 68. 7 g/kg, respectively (once per day). Rats in the MTX group were administered with MTX suspension (0.78 mg/kg) by gastrogavage, once per week for 30 successive days. The paw swelling was evaluated using volume method (draining volume). PBMCs were extrac- ted from each group after intervention. mRNA expression levels of DNMTs (DNMT1 , DNMT3a, DNMT3b) were detected by real-time quantitative PCR. Results Compared with the normal group, the paws were obviously swollen in the model group (P <0. 01). Compared with the model group, swollen paws were obviously alleviated in low, middle, and high dose WJR groups, and the MTX group (P <0.01). Compared with before treatment in the same group, swollen paws were obviously alleviated in low, middle, and high dose WJR groups, and the MTX group (P <0. 01 ). Compared with the normal group, expression levels of DNMT1, DNMT3a, and DNMT3b in PBMCs were obviously lowered in the model group (P <0.01). Compared with the model group, expression levels of DNMT1 , DNMT3a, DNMT3b in PBMCs were obviously elevated in low, middle, and high dose WJR groups, and the MTX group (all P <0. 01). There was no sig- nificant difference in expression levels of DNMT1, DNMT3a, or DNMT3b in PBMCs among low, middle, and high dose WJR groups (P>0.05). Conclusions Expression levels of DNMTs in PBMCs of CIA rats decreased. WJR up-regulated the expression level of DNMTs in PBMCs of CIA rats in no obvious dose de- pendent way. One of WJR's mechanisms for treating CIA might be up-regulating expression levels of DN- MTs, and adjusting the state of DNA methylation.

NADPH oxidase-dependent formation of reactive oxygen species contributes to transforming growth factor ß1-induced epithelial-mesenchymal transition in rat peritoneal mesothelial cells, and the role of astragalus intervention.

OBJECTIVE: To investigate the role of nicotinamide-adenine dinucleotide phosphate (NADPH) oxidasedependent formation of reactive oxygen species (ROS) in the transforming growth factor ß1 (TGF-ß1)-induced epithelial-mesenchymal transition (EMT) in rat peritoneal mesothelial cells (RPMCs), and the effect of Astragalus injection (AGI) intervention. METHODS: Primary RPMCs were cultured to the second generation in vitro. After synchronization for 24 h, the cells were randomly assigned to the following groups: control (Group A), AGI (2 g/mL; Group B), TGF-ß1 (10 ng/mL; Group C), TGF-ß1 (10 ng/mL) + AGI (2 g/mL; Group D; pretreated for 1 h with AGI before TGF-ß1 stimulation). Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis were employed to evaluate the mRNA and protein expression of the NADPH oxidase subunit p67phox, α-smooth muscle actin (α-SMA) and E-cadherin. The dichlorofluorescein-sensitive cellular ROS levels were measured by a fluorometric assay and confocal microscopy. RESULTS: TGF-ß1 significantly induced NADPH oxidase subunit p67phox mRNA and protein expression in RPMCs, as well as inducing the production of intracellular ROS. AGI inhibited this TGF-ß1-induced up-regulation by 39.3% and 47.8%, respectively (P<0.05), as well as inhibiting the TGF-ß1-induced ROS generation by 56.3% (P<0.05). TGF-ß1 also induced α-SMA mRNA and protein expression, and down-regulated E-cadherin mRNA and protein expression (P<0.05). This effect was suppressed by AGI (P<0.05). CONCLUSIONS: NADPH oxidase-dependent formation of ROS may mediate the TGF-ß1-dependent EMT in RPMCs. AGI could inhibit this process, providing a theoretical basis for AGI in the prevention of peritoneal fibrosis.

[Effects of acupoint-embedment of medicated-thread and acupoint-injection on expression of cortical urokinase-type plasminogen activator and plasminogen activator inhibitor-1 in rats with cerebral ischemia-reperfusion injury].

OBJECTIVE: To observe the effect of acupoint-embedement of medicated-thread and acupoint-injection of Chuanxiongzine on the expression of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) in the cerebral cortex in rats with cerebral ischemia-reperfusion injury (CI/RI), so as to explore its underlying mechanism in protecting the ischemic cerebral tissue. METHODS: Seventy-eight SD rats were randomly divided into normal control group (n=6), sham operation (sham) group (n=18), model group (n=18),acupoint injection (Al) group (n=18), and acupoint-thread-embedment (ATE) group (n=18). Rats of the latter 4 groups were randomized into 1 d, 3 d and 5 d subgroups, with 6 rats in each. CI/RI model was established by occlusion of the right middle cerebral artery (MCAO) for 30 min and reperfusion. For rats of the Al group, Chuanxiongzine (0.1 mL/200 g) was injected into "Baihui" (GV 20) and "Dazhui" (GV 14), and for those of ATE group, a piece of medicated thread containing collagen protein (extracted from the rat's tail tissue) and Chuanxiongzine + retarder was embedded into GV 20 and GV 14, respectively. The expression of uPA and PAI-1 in the cerebral cortex on the ischemia side was detected by immunohistochemistry. RESULTS: In comparison with the normal control group, the expression of uPA of the ischemia cerebral cortex on day 1, 3 and day 5 in the model group was increased significantly (P < 0.01), while the PAI-1 expression decreased remarkably in the model group (P < 0.01). Compared with the 3 time-points of the model group, cortical uPA expression levels at the 3 time-points in the Al group and those of day 3 and day 5 in the ATE group were down-regulated significantly (P < 0.01), whereas cortical PAI-1 expression levels at the 3 time-points in both AI and ATE groups up-regulated considerably (P < 0.05, P < 0.01). Comparison between AI and ATE groups showed that the expression levels of cortical uPA in the latter group on day 3 and day 5 were significantly lower than those of the former group (P < 0.05), whereas the cortical PAI-1 expression levels in the latter group on day 3 and day 5 were evidently higher than those of the former group (P < 0.05). But, cortical PAI-1 expression of the ATE group on day 1 was significantly lower than that of the AI group (P < 0.05). No significant differences were found between the AI and ATE groups in the expression level of cortical uPA on day 1 and between normal and sham groups in both uPA and PAI-1 expression levels at the 3 time-points (P > 0.05). CONCLUSION: Both AI and ATE can down-regulate cortical uPA expression and up-regulate cortical PAI-1 expression in rats with CI/RI, which may contribute to their protective effect in reducing cerebral ischemic injury.

[Subcellular mimical localization of the ATP synthase B subunit from Clonorchis sinensis under different conditions of cell cycling].

OBJECTIVE: To investigate the subcellular localization of ATP synthase b subunit from Clonorchis sinensis under different conditions of Hela cell cycling, and the effect of this protein on the expression of its encoding-gene and homologous host genes. METHODS: pEGFP-N1-CsATP-synt_B and the vector pEGFP-N1 were transfected into Hela cells, respectively. Transfected cells were synchronized in G0/G1 by serum starvation, G1/S, S cells by double thymidine block, and G2/M cells by thymidine-Nocodozale block. After synchronization, the subcellular localization of the expressed fusion protein was observed with a laser confocal microscope. The expression level of this fusion protein in cells was detected by flow cytometry (FCM). The expression of CsATP-synt_B and HomoATP-synt_B in different cell cycle phases accessed by RT-PCR. RESULTS: FCM results indicated that in the G0/G1 phase the expression of pEGFP-N1 vector was decreased significantly, while pEGFP-N1-CsATP-synt_B expression showed an upward trend. In the other phases of cell cycle, the protein expression was similar in the above two kinds of plasmids. The intact CsATP-synt_B was expressed in mitochondria in the G0/G1, S, and G2/M phases and nucleus during G1/S phase. After the fusion proteins entered the nucleus, the mRNA expression of CsATP-synt_B and HomoATP-synt_B increased significantly. CONCLUSION: CsATP-synt_B can be expressed in the nucleus during G1/S phase, and regulated by the cell cycle and energy requirements.

[Tissular and subcellular localization of the ATP synthase B subunit in Clonorchis sinensis].

OBJECTIVE: To illustrate the distribution of ATP synthase b subunit in the tissue of Clonorchis sinensis adult and its subcellular mimical localization in HeLa cells. METHODS: With the antiserum against recombinant CsATP-synt_B protein raised from SD rats as primary antibody, paraffin sections of the adult of C. sinensis were processed by the method of fluorescent immunohistochemistry to observe the distribution of CsATP-synt_B protein in adult worm. According to the prediction by bioinformatics of the definite mitochondrial targeting sequence (MTS) and probable Bipartite nuclear localization signals (NLS_BP)in CsATP-syntB sequence, recombinant pEGFP-N1 plasmids containing the intact and three defective CsATP-synt_B sequence with single defect of MTS or NLS_BP or double defect respectively were constructed. The recombinant plasmids and the control plasmid-pEGFP-N1, pEYFP-Mito and H2B-CFP, were transfected into the HeLa cells by Lipofectamine 2000 reagent and the subcellular location of the GFP fusion protein was observed with confocal microscopy. RESULTS: The CsATP-synt_B protein appeared to distribute all over the adult worm, especially abundant on the acetabulum, ovary, vitellarium and tegument. The intact CsATP-synt_B was definitely expressed in mitochondria and/or nucleus of infected HeLa cells, whereas the MTS-deleted mutant only in cytoplasma and nucleus, the NLS_BP-deleted mutant in mitochondria and cytoplasm, and the double defect mutant only in cytoplasm. CONCLUSION: The distribution of CsATP-synt_B in adult is accord with that of mitochondria, and mainly exits in the organs and the tissues of active energy metabolism. This study first predicted and confirmed that CsATP-synt_B can be expressed in the nucleus.

[The effects of Lysophospholipase from Clonorchis sinensis on the hepatic stellate cells and oval cells of rat].

AIM: To clarify the effects of the recombinant protein of Lysophospholipase from Clonorchis sinensis (CsLysoPLA) on the hepatic stellate cells (HSC) and oval cells of rat. METHODS: Binding of the recombinant CslysoPLA protein to the membrane of HSC and oval cells was identified by immunofluorescent staining. The HSC and oval cells were cultured and treated with the recombinant protein at different doses, and proliferation was quantified by MTT method. Cell cycle analysis was performed by flow cytometry. RESULTS: The recombinant CslysoPLA protein could bind to the membrane of HSC and oval cells. Compared to control, 2 mg/L and 20 mg/L the recombinant protein could promote HSC and oval cells growth (P<0.05), whereas 200 mg/L the recombinant protein could induce the cells necrosis, which associated with overt plasma membrane disruption. Oval cell number in G(2) phase of the recombinant protein 20 mg/L treated group was higher than that of control group. CONCLUSION: In vitro, the recombinant protein could induce HSC and oval cells proliferation at low concentrations (2 mg/L and 20 mg/L), whereas it also could induce the cells necrosis at high concentration (200 mg/L). These results suggested that CslysoPLA might play a role in the pathogenicity of C. sinensis.

[Cloning and expression of the F0-ATP synthase B chain of Clonorchis sinensis and immunogenicity identification of the recombinant protein].

OBJECTIVE: To clone and express the Clonorchis sinensis F0-ATP synthase b chain (CsF0-ATP-synt_B) gene and analyze immunogenicity of the recombinant protein. METHODS: The coding region F0-ATP synthase b chain gene with the mitochondrial targeting sequence (MTS) removed was amplified with PCR using the cloned plasmid as template, and the product was cloned into the prokaryotic expression vector pET-28a(+), transformed into E. coli BL21 (DE3) and induced with IPTG. The expressed product was purified by Ni-IDA affinity chromatography,and analyzed by SDS-PAGE for its expression and identified by Western blotting for its immunogenicity. RESULTS: The coding sequence of the F0-ATP synthase b-chain like gene removed off the MTS contains 813 base pairs encoding 271 amino acids with a theoretical molecular weight of 31,171.9. PCR, double enzyme digestion and DNA sequencing confirmed that the recombinant plasmid pET-28a (+)-CsF0-ATP-synt_B was constructed successfully, and the resolvable expression was obtained in E.coli BL21. Highly purified recombinant protein was prepared through affinity chromatography. The recombinant protein could be recognized by the immune serum of the SD rat immunized with the recombinant protein. CONCLUSION: The CsF0-ATP-synt_B like gene has been efficiently expressed in prokaryotic expression system with immunogenicity.

[Construction and characterization of a cDNA library from human liver tissue of cirrhosis].

OBJECTIVE: To construct a cDNA library from human liver tissue of cirrhosis. METHODS: The total RNA from human liver tissue of cirrhosis was extracted using Trizol method, and the mRNA was purified using mRNA purification kit. SMART technique and CDSIII/3' primer were used for first-strand cDNA synthesis. Long distance PCR was then used to synthesize the double-strand cDNA that was then digested by proteinase K and Sfi I, and was fractionated by CHOMA SPIN-400 column. The cDNA fragments longer than 0.4 kb were collected and ligated to lambdaTripl Ex2 vector. Then lambda-phage packaging reaction and library amplification were performed. The qualities of both unamplified and amplified cDNA libraries was strictly checked by conventional titer determination. Eleven plaques were randomly picked and tested using PCR with universal primers derived from the sequence flanking the vector. RESULTS: The titers of unamplifed and amplified libraries were 1.03 x 10(6) pfu/ml and 1.36 x 10(9) pfu/ml respectively. The percentages of recombinants from both libraries were 97.24 % in unamplified library and 99.02 % in amplified library. The lengths of the inserts were 1.02 kb in average (36.36 % 1 approximately equals 2 kb and 63.64 % 0.5 approximately equals 1.0 kb). CONCLUSION: A high quality cDNA library from human liver tissue of cirrhosis was constructed successfully, which can be used for screening and cloning new special genes associated with the occurrence of cirrhosis.

Construction and characterization of a cDNA library from human liver tissue with chronic hepatitis B.

OBJECTIVE: To construct a cDNA library from human liver tissue with chronic hepatitis B and check its quality for investigating the expression level of liver tissue infected by hepatitis B virus. This will then be used to find the relevant genes and interesting proteins associated with the development of hepatitis B. METHODS: The total RNA from liver tissue with chronic hepatitis B was extracted and the mRNA was purified using TRIZOL method. Switching mechanism at 5' end of the RNA transcript (SMART) technique and CDS III/3' primer were used for first-strand cDNA synthesis. Long distance polymerase chain reaction (LD PCR) was then used to synthesize the double-strand cDNA that was then digested by Sfi I and fractionated by CHROMA SPIN-400 column. The longer than 0.4 kb cDNAs were collected and ligated to lambdaTriplEx2 vector. Then lambda phage packaging reaction and library amplification were performed. The qualities of both unamplified and amplified cDNA libraries were strictly checked by conventional titer determination. Fourteen plaques were randomly picked and tested using PCR with universal primers derived from the sequence flanking the vector. RESULTS: The titers of unamplifed and amplified libraries were 1.94 x 10(6) pfu/ml and 1.49 x 10(9) pfu/ml respectively. The percentages of recombinants from both libraries were 98.15% in unamplified library and 98.76% in amplified library. The lengths of the inserts were 1.23 kb in average, 1-2 kb in 64.29%, and 0.5-1.0 kb in 35.71%. CONCLUSION: A high quality cDNA library from human liver tissue with chronic hepatitis B was successfully constructed.