Mechanism of Polyphyllin Ⅰ Extract in Activating Hippo Signal to Induce Apoptosis and Autophagy of Colorectal Cancer Cells / 中国实验方剂学杂志

ObjectiveTo study the inhibitory effect of polyphyllin Ⅰ （PPI） on the growth of colorectal cancer cells and its molecular mechanism. MethodRKO cells were cultured and divided into a blank group and PPI treatment groups with concentrations of 0.6， 0.8， 1.0 μmol·L-1， respectively. HRT18 cells were cultured and divided into a blank group and PPI treatment groups with concentrations of 1.2， 1.4， 1.6 μmol·L-1， respectively. The effects of PPI on the proliferation and morphology of colorectal cancer were detected by cell proliferation toxicity assay， trypan blue exclusion assay， plate clone formation assay， and confocal high-intension cell imaging analysis system. Flow cytometry was used to detect the apoptosis rate of colorectal cancer cells. The pQCXIP-GFP-LC3 plasmid transfection assay was used to detect the formation of autophagosomes in colorectal cancer cells after PPI treatment. Western blot was used to detect the expression of apoptosis-related proteins Caspase-3， Caspase-8， and poly ADP ribose polymerase （PARP）， the expression of autophagy related protein LC3Ⅱ， and the expression and phosphorylation of Hippo signaling pathway proteins LATS1 and YAP. In the plvx-Flag-YAP plasmid transfection assay， YAP was overexpressed and treated with PPI， and the proliferation of colorectal cancer cells was detected by cytotoxicity assay. The expression of LC3Ⅱ and PARP in colorectal cancer cells was detected by Western blot. SwissADME predicted pharmacokinetic parameters of PPI. ResultAs compared with the blank group， the survival rate and clone formation ability of colorectal cancer cells in the PPI group were significantly decreased （P<0.01）， the cell area of colorectal cancer cells in the PPI group was significantly decreased， and the roundness of colorectal cancer cells was significantly increased （P<0.01）. As compared with the blank group， the apoptosis rate of colorectal cancer cells in PPI treatment groupw was significantly increased （P<0.01）， the expression of apoptotic proteins Caspase-3 and Caspase-8 protein precursor in PPI treatment groups was decreased， and the cleavage of PARP was increased （P<0.01）. As compared with the blank group， the expression level of autophagy-related protein LC3Ⅱ in colorectal cancer cells in PPI treatment groups was significantly increased， and the formation of autophagosomes was promoted （P<0.01）. As compared with the blank group， the expression of YAP protein in colorectal cancer cells in PPI treatment groups was significantly decreased， and the expressions of phosphorylated LATS1 and YAP were significantly increased （P<0.01）. As compared with the blank group， overexpression of YAP could significantly antagonize the effect of PPI on apoptosis， autophagy activation， and proliferation inhibition of colorectal cancer cells. SwissADME simulation results showed that PPI had good drug like activity. ConclusionPPI can induce apoptosis and autophagy of colorectal cancer cells through targeted activation of Hippo signaling pathway， thereby inhibiting their proliferation.

Hydnocarpin inhibits malignant progression of triple negative breast cancer via CNOT4-mediated ubiquitination and degradation of YAP / 中国中药杂志

This study aims to investigate the effect and mechanism of hydnocarpin(HC) in treating triple negative breast cancer(TNBC). Cell counting kit-8(CCK-8), xCELLigence real-time cellular analysis(RTCA), and colony formation assay were employed to determine the effects of HC on the proliferation of two TNBC cell lines: MDA-MB-231 and MDA-MB-436. The effects of HC on the migration and invasion of TNBC cells were detected by high-content analysis, wound-healing assay, and Transwell assay. The changes in the epithelial-mesenchymal transition(EMT) and the expression of invasion-and migration-associated proteins [E-cadherin, vimentin, Snail, matrix metalloproteinase-2(MMP-2), and MMP-9] were detected by Western blot. Western blot and RT-qPCR were employed to determine the protein and mRNA levels of Yes-associated protein(YAP) and downstream targets(CTGF and Cyr61). TNBC cells were transfected with Flag-YAP for the overexpression of YAP, and the role of YAP as a key target for HC to inhibit TNBC malignant progression was examined by CCK-8 assay, Transwell assay, and wound-healing assay. The pathway of HC-induced YAP degradation was detected by the co-treatment of proteasome inhibitor with HC and ubiquitination assay. The binding of HC to YAP and the E3 ubiquitin ligase Ccr4-not transcription complex subunit 4(CNOT4) was detected by microscale thermophoresis(MST) assay and drug affinity responsive target stability(DARTS) assay. The results showed that HC significantly inhibited the proliferation, colony formation, invasion, and EMT of TNBC cells. HC down-regulated the protein and mRNA levels of CTGF and Cyr61. HC down-regulated the total protein level of YAP, while it had no effect on the mRNA level of YAP. The overexpression of YAP antagonized the inhibitory effects of HC on the proliferation, migration, and invasion of TNBC cells. HC promoted the degradation of YAP through the proteasome pathway and up-regulated the ubiquitination level of YAP. The results of MST and DARTS demonstrated direct binding between HC, YAP, and CNOT4. The above results indicated that HC inhibited the malignant progression of TNBC via CNOT4-mediated degradation and ubiquitination of YAP.

Mechanism of polyphyllin A in inhibition of proliferation and induction of apoptosis of gastric cancer cells by directly targeting ETS-1 / 中国中药杂志

The present study investigated the mechanism of polyphyllin A(PPA) in inhibiting gastric cancer(GC) cells. GC cells(SGC7901 and MGC803 cell lines) were treated with PPA at different concentrations. The effect of PPA on the proliferation of GC cells was detected by MTT assay, real-time cell analysis(RTCA) assay, and clone-forming assay, respectively. Reactive oxygen species(ROS) of GC cells was detected by flow cytometry. The change of mitochondrial membrane potential was detected by JC-1 assay. The expression and phosphorylation levels of apoptosis-related proteins(caspase-9, caspase-3, and PARP) and proteins related to the signaling pathway(ETS-1, CIP2 A, and Akt) were detected by Western blot. The binding sites of PPA to ETS-1 were analyzed by molecular docking. The affinity of PPA and ETS-1 was detected by drug affinity responsive target stability(DARTS) assay. PPA had a significant inhibitory effect on the proliferation and colony formation of GC cells at a low concentration. The PPA groups showed increased ROS and decreased mitochondrial membrane potential. PPA down-regulated the precursor expression of caspase-9 and caspase-3 and promoted the cleavage of PARP, suggesting that PPA induced the apoptosis of GC cells through the mitochondrial pathway. PPA significantly reduced expression levels of CIP2 A and the phosphorylation of downstream Akt. Molecular docking showed that PPA bound to the ETS domain of ETS-1, the transcription factor of CIP2 A, and formed hydrogen bonds with Pro319 and Asp317. DARTS assay further confirmed that PPA significantly prevented the hydrolysis of ETS-1 by pronase, which was inductive of the direct binding effect of PPA and ETS-1. PPA inhibits the proliferation and induces the apoptosis of GC cells by directly targeting ETS-1 to down-regulate the ETS-1/CIP2 A/Akt signaling pathway.

Paris saponin VII, a direct activator of AMPK, induces autophagy and exhibits therapeutic potential in non-small-cell lung cancer / 中国天然药物

Paris saponin VII (PSVII), a bioactive constituent extracted from Trillium tschonoskii Maxim., is cytotoxic to several cancer types. This study was designed to explore whether PSVII prevents non-small-cell lung cancer (NSCLC) proliferation and to investigate its molecular target. AMP-activated protein kinase (AMPK) has been implicated in the activation of autophagy in distinct tissues. In cultured human NSCLC cell lines, PSVII induces autophagy by activating AMPK and inhibiting mTOR signaling. Furthermore, PSVII-induced autophagy activation was reversed by the AMPK inhibitor compound C. Computational docking analysis showed that PSVII directly interacted with the allosteric drug and metabolite site of AMPK to stabilize its activation. Microscale thermophoresis assay and drug affinity responsive target stability assay further confirmed the high affinity between PSVII and AMPK. In summary, PSVII acts as a direct AMPK activator to induce cell autophagy, which inhibits the growth of NSCLC cells. In the future, PSVII therapy should be applied to treat patients with NSCLC.

Comparative Study of Longbie Capsules and Bone Marrow Mesenchymal Stem Cell Transplantation in Repairing Damaged Cartilage of Knee Osteoarthritis Rats / 广州中医药大学学报

Objective To compare the actions of Longbie Capsules and bone marrow mesenchymal stem cell (BMSC)transplantation in repairing the damaged cartilage of knee osteoarthritis(KOA)rats. Methods Thirty-six rats aged 4-6 weeks old were induced into KOA model(bilateral knees)by collagenase injection method. All of the modeled rats were randomly divided into model group(intragastric administration of normal saline), BMSC transplantation group(giving tail vein injection of 1 ×106 of BMSCs per time, 2 times every week), and Chinese medicine group (intragastric administration of Longbie Capsules of 7.5 g·kg-1·d-1),12 rats in each group. Six weeks later,the cartilage of rat bilateral knees was taken out. The pathological changes of cartilage were observed by hematoxylin-eosin(HE) staining method, and the protein and mRNA expression levels of Col2a1, X-linkedinhibitor of apoptosis protein (XIAP), HuR in rat knee cartilage were detected by immunohistochemistry and real-time quantitative polymerase chain reaction (qPCR), respectively. Results The HE staining results showed that the cartilage tissue surface was rough with more cracks, and the cartilage cells gathered with the cytoplasm collapsed and arranging disorderly in the model group. The number of chondrocytes was increased and cell surface was flat,and the cracks of the cartilage were decreased with the chondrocytes arranging uniformly in Chinese medicine group and BMSC transplantation group compared with the model group. The results of immunohistochemistry and qPCR detection showed that in Chinese medicine dosage group and BMSC transplantation group, the protein and mRNA expression levels of Col2a1,XIAP and HuR were significantly higher than those in the model group (P<0.05 or P<0.0001), but there was no significant difference between the two medication groups(P>0.05). Conclusion Longbie Capsules and BMSC transplantation can promote the secretion of Col2a1 in the cartilage tissue of KOA rats,improve cartilage, and their mechanism may be related with up-regulating apoptosis-related proteins HuR and XIAP.

Allogeneic demineralized cancellous bone for cartilage tissue engineering / 西安交通大学学报(医学版)

Objective Chondrocytes were seeded on allogeneic demineralized cancellous bone (DCB ) constructing tissue-engineered cartilage to evaluate the feasibility of DCB as scaffolds for tissue engineering cartilage.Methods Allogeneic DCB was prepared by successive defatting and decalcification and then observed under scanning electronic microscope.One-month old rabbit articular chondrocytes were isolated and proliferated by monolayer culture.Passage 1 chondrocytes were seeded on DCB to construct tissue-engineered cartilage in vitro for 6 weeks.Specimens were taken after 7,14,21,28,35 and 42 days'culture and evaluated by hematoxylin and eosin (HE),toluidine blue (TB),and immunohistochemistry for collagen type Ⅰ and Ⅱ.Results Prepared DCB showed three-dimensional porous structure of 100 - 500 μm with good plasticity and certain mechanical strength. Chondrocytes grew well on and inside the DCB,and were located in lacunae and expressed matrix proteoglycan and type collagen Ⅱ,which formed cartiageous tissues on DCB up to 42 days'culture.Conclusion DCB combined with allogeneic chondrocytes successfully constructed tissue-engineered cartilage in vitro ,which is an ideal scaffold for tissue engineering cartilage.

Allogeneic demineralized cancellous bone for cartilage tissue engineering / 西安交通大学学报(医学版)

Objective Chondrocytes were seeded on allogeneic demineralized cancellous bone (DCB ) constructing tissue-engineered cartilage to evaluate the feasibility of DCB as scaffolds for tissue engineering cartilage.Methods Allogeneic DCB was prepared by successive defatting and decalcification and then observed under scanning electronic microscope.One-month old rabbit articular chondrocytes were isolated and proliferated by monolayer culture.Passage 1 chondrocytes were seeded on DCB to construct tissue-engineered cartilage in vitro for 6 weeks.Specimens were taken after 7,14,21,28,35 and 42 days'culture and evaluated by hematoxylin and eosin (HE),toluidine blue (TB),and immunohistochemistry for collagen type Ⅰ and Ⅱ.Results Prepared DCB showed three-dimensional porous structure of 100 - 500 μm with good plasticity and certain mechanical strength. Chondrocytes grew well on and inside the DCB,and were located in lacunae and expressed matrix proteoglycan and type collagen Ⅱ,which formed cartiageous tissues on DCB up to 42 days'culture.Conclusion DCB combined with allogeneic chondrocytes successfully constructed tissue-engineered cartilage in vitro ,which is an ideal scaffold for tissue engineering cartilage.

Key technologies of clinical study of traditional Chinese medicine on pediatric functional abdominal pain / 药物评价研究

Functional abdominal pain is one of the most common problems in functional gastrointestinal disorders,and it's also one of the diseases benefit most from traditional Chinese medicine (TCM) treatment.This paper illustrates some key considerations on the study design of traditional Chinese medicine intended for the treatment of FAP based on the latest treatment and evaluation guidelines,technical guidance,professional authority works as well as the latest clinical studies,and combined with experience of clinical trial design.It hopes to offer helps for research designers in this genera.

The lived experience of family members caring for schizophrenia patients at home:a systematic review of qualitative studies / 中华护理杂志

Objective To systematically review and synthesize the lived experience of family members caring for schizophrenia patients at home,in order to provide evidence for community and home nursing. Methods We searched databases including The Cochrane Library,PubMed,EMbase,ISI Web of Science,PsycINFO,CINAHL,CBM, CNKI,VIP and Wanfang from inception to April 2017,to collect qualitative studies in the experience of family members caring for schizophrenia patients at home. The quality of included studies was evaluated according to JBI Critical Appraisal Tool for qualitative studies in Australia. Results A total of 31 studies were included,and 141 complete findings were grouped according to their similarities to form 8 categories. These categories resulted in two synthesized findings:Integration Results 1:It brought family members a negative influence in care process because of excessive pressure and burden,but over time,they were slowly accepting the fact and trying to cope with dis-ease;Integration Results 2:Patients were unable to take care of themselves,and caregivers were helpless and wanted assistance from the government and the health care system. Conclusion The government and health system should pay more attention to the impact of schizophrenia on family members who take care of schizophrenia patients. In the process of care,patients should be given support,guidance and encouragement,which help family members to improve coping abilities of psychology and disease,and to promote physical and mental health of schizophrenia pa-tients and their family members.

Relationship between serum level of caveolin-1 and early neurological deterioration in patients with acute cerebral infarction / 上海交通大学学报（医学版）

Objective·To investigate the relationship between serum level of caveolin-1 (Cav-1) and early neurological deterioration (END) in patients with acute cerebral infarction. Methods·A total of 126 consecutive patients with acute cerebral infarction were recruited from July 2016 to January 2017 in Department of Neurology, the First Affiliated Hospital of Chongqing Medical University. The serum Cav-1 levels of all patients were detected by enzyme-linked immunosorbent assay (ELISA) test. The neurological deficits were assessed by the National Institutes of Health Stroke Scale (NIHSS) and the Glasgow Coma Scale (GCS) at the same time. Compared with the admission baseline NIHSS score, if second motor NIHSS score increased ≥ 1 point or the total NIHSS score increased ≥ 2 points within 3 days after hospitalization, they were classified as END group, otherwise they were classified as non-END group. Multivariable Logistic regression analysis was used to examine the independent predictors of END in the patients. Receiver operating characteristic (ROC) curves were obtained to explore Cav-1 levels in predicting END. Results·Serum Cav-1 levels in END group were significantly higher than those in non-END group [(29.88±19.57) ng/mL vs (16.08±13.37) ng/mL, P=0.000]. Based on the ROC curves, the best cut-off point of serum Cav-1 for predicting END was 16.55 ng/mL. The sensitivity and specificity were 73.33％ and 74.07％, respectively. Multivariable Logistic regression analysis showed that Cav-1≥16.55 ng/mL remained an independent predictor of END (OR=4.936, 95％CI 1.608-15.155, P=0.005). Conclusion·Serum Cav-1 is an independent predictor of END in patients with acute cerebral infarction.

Genetic Characteristics of Coxsackievirus Group A Type 4 Isolated from Patients with Acute Flaccid Paralysis in Shaanxi, China / 病毒学报

We analyzed the genetic characteristics of coxsackievirus A4 (CV-A4) based on the entire VP1 coding region. Samples were isolated from patients with acute flaccid paralysis (AFP) in Shaanxi, China from 2006 to 2010. We wished to ascertain the predominant genotype and the relationship between CV-A4 infection and AFP. Sixty-eight non-polio enteroviruses were inoculated onto RD cells (to increase the virus titer) and molecular typing was undertaken. The entire VP1 coding region was amplified. Percentage of CV-A4 was 10.3% (7/68). Analyses of genetic identify and creation of phylogenetic trees revealed that CV-A4 could be classified into A, B and C genotypes. Seven CV-A4 strains from Shaanxi and other CV-A4 strains from China formed an independent evolution lineage located in group 4 and belonged to the C2 sub-genotype. These data suggested that CV-A4 strains of sub-genotype C2 were the predominant genotypes in China. These strains co-evolved and co-circulated with those from other provinces in China, so continued monitoring of CV-A4 (by clinical and genetic surveillance) should be enhanced.

Clinical Etiologies of Fever of Unknown Origin in 500 Cases / 中国医学科学院学报

<p><b>OBJECTIVE</b>To investigate the distribution and change of the causes of fever of unknown origin(FUO).</p><p><b>METHODS</b>The clinical data of 500 inpatients with FUO in our center between December 2003 and June 2014 were retrospectively analyzed. The diagnostic methods,etiologies,and their possible relationship with age,sex,fever duration,and period.</p><p><b>RESULTS</b>Of these 500 FUO patients,452(90.4%)were confirmed to be with fever caused by conditions including infectious diseases [(n=231,46.2%;e.g.tuberculosis(32.9%,76/231)],connective tissue diseases(CTD)(n=99,19.8%),neoplasms(n=58,11.6%),miscellaneous causes(n=64,12.8%). The causes were not identified in 48 cases(9.6%).The proportion of CTD in female patients was significantly higher than that in male patients(26.3% vs. 14.5%,P=0.025),whereas the proportion of neoplasms in male patients was significantly higher than that in female patients(14.5% vs. 8.0%,P=0.001). Infectious diseases was the most common cause in all age groups,CTD ranked the second in the 21-39-year group and 40-59-year group,and neoplasm was the second most coomon cause in the over 60 year group. Thus,the distribution of FUO etiologies significantly differed in different age groups(χ(2)=43.10,P=0.000). The duration of fever in patients with neoplasms [60(28,90)d] was longer than that in patients with infectious diseases [28(21,42)d,Z=-4.168,P=0.000] or CTD [30(21,60)d,Z=-2.406,P=0.016)]. Compared with the level in 2003-2008,the proportion of CTD significantly increased in 2009-2014(13.7% vs. 23.8%,χ(2)=8.598,P=0.003),along with the dicrease of the proportions of infectious diseases,neoplasms and miscellaneous diseases were decreased(all P>0.05).</p><p><b>CONCLUSIONS</b>Infectious diseases(in particular,tuberculosis)remains the major cause of FUO. CTD and neoplasms also play important roles in the development of FUO. The distributions of the FUO etiologies have certain differences in terms of age,sex,duration of fever,and period.</p>

Effect of 18F-2-deoxy-2-fluoro-d-glucose on proliferation of Lewis lung cancer cell / 中华放射医学与防护杂志

Objective To investigate the influence of 18F-FDG on the proliferation of Lewis lung cancer cell line,and to elucidate its possible mechanism.Methods Morphological changes of cells after culture for 24 h at different concentrations of 0,0.37,1.85,3.70 and 7.4 (×106) Bq/ml of 18F-FDG were observed by using inverted microscopy and electron microscopy.The apoptosis and phase distribution of cell cycle of irradiated cells were analyzed with flow cytometry.DNA synthesis of irradiated cells was assayed by 3H-TdR incorporation.Lipid peroxidation was measured by chromometry and expression of Bcl-2 and Bax protein was measured by immunohistochemical technique.Results Exposed to (0-7.40) × 106Bq/ml of 18F-FDG for 24 h,the cumulative absorbed doses delivered to cells in five groups were 0,0.11,0.55,1.10 and 2.20 Gy,respectively.Irradiated cells showed morphological changes of apoptosis.The apoptosis rate of irradiated cells was increased from (4.05 ± 0.01)% to (25.6 ± 0.28) % (t = 188,P＜0.01).3H-TdR incorporation rate was decreased from 100% to(22.0 ± 0.51)% (t =27.6,P ＜0.05).The levels of M DA in cells were augmented from (0.08 ± 0.03) to (0.67 ± 0.12) μmol/L (t =11.7,P ＜ 0.01).Cell cycle arrest was found in G2/M phase with the increasing doses from 0 to 2.20 Gy.The expression of Bcl-2 protein was decreased while that of Bax protein increased.Conclusions 18F-FDG could induce the apoptosis of cells and inhibit the proliferation of cells.

Study on a high coverage of measles vaccine while high incidence of measles disease still appeared in Shaanxi province / 中华流行病学杂志

<p><b>OBJECTIVE</b>To analyze the affecting factors on the cause of measles and measles vaccine under the high coverage of measles vaccine in Shaanxi province.</p><p><b>METHODS</b>Age distribution and vaccination history on measles cases were studied. Throat swabs were obtained from measles cases. Measles virus was isolated from collected specimens with phenol-chloroform extraction method. Amplification was performed by RT-PCR in order to amplify 450 bp fragment of the -COOH side of N gene,and then the sequences of PCR products were detected to confirm the gene type of measles virus. Sera were obtained from patients who were in acute phase of measles disease,and antibody titer against measles vaccine strain and wild strain were determined by small amounts neutralization test.</p><p><b>RESULTS</b>Measles cases with the history of measles vaccination were accounted for 38.97% of the total numbers. The geometrical mean titer (GMT) (56.18) against S191 attenuated strain was significant higher than that of wild strain (26.90) among these measles patients with history of having received measles vaccination. The GMT (25.40) against S191 attenuated strain was similar to that of wild strain (27.86) among these measles patients with non-history of measles vaccination. The antibody negative rate against wild strain was 19.15% to these sera from patients with the history of measles vaccination and antibody potency against S191 strain was less than 16.</p><p><b>CONCLUSION</b>The appearance of higher measles incidence under the higher coverage of measles vaccine indicated that measles epidemic strain might degenerate as the result regarding the failure of the present measles vaccine in protecting the transmission of H1 wild strain.</p>