Phillygenin inhibited M1 macrophage polarization and reduced hepatic stellate cell activation by inhibiting macrophage exosomal miR-125b-5p.

Liver fibrosis (LF) is an important stage in chronic liver disease development, characterized by hepatic stellate cell (HSC) activation and excessive extracellular matrix deposition. Phillygenin (PHI), an active component in the traditional Chinese medicine Forsythiae Fructus with a significant anti-inflammatory effect, has been proved to inhibit HSC activation. Macrophages can polarize to pro-inflammatory M1 phenotype and anti-inflammatory M2 phenotype, participating in LF development. Currently, Forsythiae Fructus and its many components have been proved to inhibit the inflammatory activation of macrophages. However, there is no direct evidence that PHI can regulate macrophage polarization, and the relationship between macrophage polarization and the anti-LF effect of PHI has not been studied. In this study, we found that PHI inhibited the co-expression of CD80 and CD86, and inhibited the mRNA expression and protein secretion of related inflammatory cytokines in RAW264.7 cells. For mechanism, PHI was found to inhibit the JAK1/JAK2-STAT1 and Notch1 signaling pathways. Subsequently, mHSCs were co-cultured with the conditioned media or exosomes from macrophages with different treatments. It was found that the conditioned media and exosomes from PHI-treated macrophages inhibited the expression of MMP2, TIMP1, TGF-ß, α-SMA, COL1 and NF-κB in mHSCs. Moreover, through bioinformatic analysis and cell transfection, we confirmed that PHI reduced HSC activation by inhibiting the overexpression of miR-125b-5p in M1 macrophage-derived exosomes and restoring Stard13 expression in mHSCs. On the whole, PHI could inhibit M1 macrophage polarization by suppressing the JAK1/JAK2-STAT1 and Notch1 signaling pathways, and reduce HSC activation by inhibiting macrophage exosomal miR-125b-5p targeting Stard13. DATA AVAILABILITY: The raw data supporting the conclusions of this study are available in the article/Supplementary figures, and can be obtained from the first or corresponding author.

CD44-Targeting Drug Delivery System of Exosomes Loading Forsythiaside A Combats Liver Fibrosis via Regulating NLRP3-Mediated Pyroptosis.

Liver fibrosis is a progressive pathological process induced by various stimuli and may progress to liver cirrhosis and cancer. Forsythiaside A (FA) is an active ingredient extracted from traditional Chinese medicine Forsythiae Fructus and has prominent hepatoprotective activities. However, the unsatisfactory pharmacokinetic properties restrict its clinical application. In this study, the nanocarrier of CD44-specific ligand Hyaluronic acid (HA)-modified milk-derived exosomes (mExo) encapsulated with FA (HA-mExo-FA) is developed. As a result, HA modification could deliver drug-loaded exosomes to the target cells and form a specific ligand-receptor interaction with CD44, thus improving the anti-liver fibrosis effect of FA. In vitro findings indicate that HA-mExo-FA could inhibit TGF-ß1-induced LX2 cell proliferation, reduce α-SMA and collagen gene and protein levels, and promote the apoptosis of activated LX2 cells. In vivo results demonstrate that HA-mExo-FA could improve liver morphology and function changes in zebrafish larvae. The anti-liver fibrosis mechanism of HA-mExo-FA may be attributed to the inhibition of NLRP3-mediated pyroptosis. In addition, the effect of HA-mExo-FA on TAA-induced increase in NLRP3 production is attenuated by NLRP3 inhibitor MCC950. Collectively, this study demonstrates the promising application of HA-mExo-FA in drug delivery with high specificity and provides a powerful and novel delivery platform for liver fibrosis therapy.

Mesenchymal stem cell-derived exosomes and non-coding RNAs: Regulatory and therapeutic role in liver diseases.

Liver disease has become a major health problem worldwide due to its high morbidity and mortality. In recent years, a large body of literature has shown that mesenchymal stem cell-derived exosomes (MSC-Exo) are able to play similar physiological roles as mesenchymal stem cells (MSCs). More importantly, there is no immune rejection caused by transplanted cells and the risk of tumor formation, which has become a new strategy for the treatment of various liver diseases. Moreover, accumulating evidence suggests that non-coding RNAs (ncRNAs) are the main effectors by which they exert hepatoprotective effects. Therefore, by searching the databases of Web of Science, PubMed, ScienceDirect, Google Scholar and CNKI, this review comprehensively reviewed the therapeutic effects of MSC-Exo and ncRNAs in liver diseases, including liver injury, liver fibrosis, and hepatocellular carcinoma. According to the data, the therapeutic effects of MSC-Exo and ncRNAs on liver diseases are closely related to a variety of molecular mechanisms, including inhibition of inflammatory response, alleviation of liver oxidative stress, inhibition of apoptosis of hepatocytes and endothelial cells, promotion of angiogenesis, blocking the cell cycle of hepatocellular carcinoma, and inhibition of activation and proliferation of hepatic stellate cells. These important findings will provide a direction and basis for us to explore the potential of MSC-Exo and ncRNAs in the clinical treatment of liver diseases in the future.

Forsythiaside A alleviated carbon tetrachloride-induced liver fibrosis by modulating gut microbiota composition to increase short-chain fatty acids and restoring bile acids metabolism disorder.

Liver fibrosis is a chronic and progressive disease with complex pathogenesis related to bile acids (BAs) and gut microbiota. Forsythiaside A (FTA), isolated from the traditional Chinese medicine Forsythiae Fructus (Lian Qiao), is a natural hepatoprotective agent. The purpose of this study was to investigate the protective effect of FTA on carbon tetrachloride (CCl4)-induced liver fibrosis in mice. Liver fibrosis was induced in mice by intraperitoneal injection of 2 mL/kg CCl4 three times a week for 4 weeks. FTA attenuated CCl4-induced liver fibrosis in mice, which was proved by the results of Masson and Sirius red staining, liver hydroxyproline, hyaluronic acid, laminin, type III procollagen, and type IV collagen assays. FTA inhibited hepatic stellate cell activation, and reduced hepatic inflammation and oxidative stress in mice treated with CCl4. What's more, FTA ameliorated CCl4-induced gut dysbiosis, maintained intestinal barrier function, increased the production of short-chain fatty acids (SCFAs), and improved endotoxemia, as manifested by decreased serum lipopolysaccharide levels and increased expression of ileal tight junction proteins. Besides, FTA can modulate the genes related to bile acid metabolism to alter the distribution of fecal BAs in fibrotic mice. In a word, FTA can improve liver fibrosis by inhibiting inflammation and oxidative stress, regulating gut microbiota and BA metabolism, and increasing the content of SCFAs. The results of this study provided an important reference for the study on the mechanisms by which natural products prevent liver fibrosis.

A review: Pharmacology and pharmacokinetics of Schisandrin A.

Schisandrin A (SA) is a bioactive lignan isolated from the traditional Chinese medicine Fructus schisandrae chinensis. In recent years, it has attracted extensive attention because of its multiple pharmacological activities. This review is the first to provide an overview of SA-related pharmacological effects and pharmacokinetic characteristics. The results showed that SA had many pharmacological effects, such as antiinflammation, anticancer, hepatoprotection, antioxidation, neuroprotection, antidiabetes mellitus, and musculoskeletal protection. Among them, NF-κB, Nrf2, MAPK, NLRP3, PI3K/AKT, Wnt, miRNA, P-gp, CYP450, PXR, and other signal transduction pathways are involved. Pharmacokinetic studies showed that SA had good pharmacokinetic characteristics, but these were affected by other factors, such as drugs or hepatic fibrosis. Thus, SA has a variety of pharmacological effects and good pharmacokinetic characteristics, which is worthy of further research and development in the future.

Diagnostic value of colposcopy in patients with cytology-negative and HR-HPV-positive cervical lesions.

PURPOSE: High-risk human papillomavirus (HR-HPV)-positive but cytology-negative cervical cancer screening results are not uncommon. This study aimed to investigate colposcopy's accuracy and diagnostic value in patients with cytology-negative HR-HPV-positive screening results. METHODS: This retrospective study included patients with HR-HPV-positive cytology-negative screening results who underwent electronic colposcopy with acetic acid and multi-point cervical biopsy, HPV typing (24 HPV subtypes), and quantitative HPV detection. RESULTS: Among 229 patients, 130 had chronic cervicitis, and 99 had cervical lesions (CIN1, n = 37; CIN2/3, n = 55; invasive carcinoma, n = 7). Using colposcopy as a reference, the cervical cytology false-negative rate was 43.2% (99/229). Colposcopy was more accurate in patients with HR-HPV16/18 or high viral loads. Multivariable analyses showed HPV viral load and childbearing history were the independent factors affecting the accuracy of colposcopy (P < 0.05). CONCLUSION: Colposcopy in HR-HPV-positive cytology-negative patients has a moderate diagnostic accuracy. The type of cervical transformation zone and HPV viral load are independent factors affecting the accuracy of colposcopy-based diagnosis.

Macrophage Polarization and Its Role in Liver Disease.

Macrophages are important immune cells in innate immunity, and have remarkable heterogeneity and polarization. Under pathological conditions, in addition to the resident macrophages, other macrophages are also recruited to the diseased tissues, and polarize to various phenotypes (mainly M1 and M2) under the stimulation of various factors in the microenvironment, thus playing different roles and functions. Liver diseases are hepatic pathological changes caused by a variety of pathogenic factors (viruses, alcohol, drugs, etc.), including acute liver injury, viral hepatitis, alcoholic liver disease, metabolic-associated fatty liver disease, liver fibrosis, and hepatocellular carcinoma. Recent studies have shown that macrophage polarization plays an important role in the initiation and development of liver diseases. However, because both macrophage polarization and the pathogenesis of liver diseases are complex, the role and mechanism of macrophage polarization in liver diseases need to be further clarified. Therefore, the origin of hepatic macrophages, and the phenotypes and mechanisms of macrophage polarization are reviewed first in this paper. It is found that macrophage polarization involves several molecular mechanisms, mainly including TLR4/NF-κB, JAK/STATs, TGF-ß/Smads, PPARγ, Notch, and miRNA signaling pathways. In addition, this paper also expounds the role and mechanism of macrophage polarization in various liver diseases, which aims to provide references for further research of macrophage polarization in liver diseases, contributing to the therapeutic strategy of ameliorating liver diseases by modulating macrophage polarization.

The Potential Application of Chinese Medicine in Liver Diseases: A New Opportunity.

Liver diseases have been a common challenge for people all over the world, which threatens the quality of life and safety of hundreds of millions of patients. China is a major country with liver diseases. Metabolic associated fatty liver disease, hepatitis B virus and alcoholic liver disease are the three most common liver diseases in our country, and the number of patients with liver cancer is increasing. Therefore, finding effective drugs to treat liver disease has become an urgent task. Chinese medicine (CM) has the advantages of low cost, high safety, and various biological activities, which is an important factor for the prevention and treatment of liver diseases. This review systematically summarizes the potential of CM in the treatment of liver diseases, showing that CM can alleviate liver diseases by regulating lipid metabolism, bile acid metabolism, immune function, and gut microbiota, as well as exerting anti-liver injury, anti-oxidation, and anti-hepatitis virus effects. Among them, Keap1/Nrf2, TGF-ß/SMADS, p38 MAPK, NF-κB/IκBα, NF-κB-NLRP3, PI3K/Akt, TLR4-MyD88-NF-κB and IL-6/STAT3 signaling pathways are mainly involved. In conclusion, CM is very likely to be a potential candidate for liver disease treatment based on modern phytochemistry, pharmacology, and genomeproteomics, which needs more clinical trials to further clarify its importance in the treatment of liver diseases.

Significance of the viral load of high-risk HPV in the diagnosis and prediction of cervical lesions: a retrospective study.

BACKGROUND: The significance of HPV viral load in the detection of cervical lesions is still controversial. This study analyzed the correlation between the high-risk HPV viral load and different cervical lesion degrees. METHODS: This retrospective study included women positive for high-risk HPV DNA and screened for cervical lesions between 01/2015 and 06/2018. The high-risk HPV DNA load was measured by the second-generation Hybrid Capture technology and classified as low, moderate, and high. Colposcopy and biopsy were performed in all patients. The patients were grouped as normal, cervical intraepithelial neoplasia (CIN) grade 1, CIN grade 2, CIN grade 3, and cervical cancer. Multivariable logistic regression was performed to explore the association between high-risk HPV DNA load and cervical lesions. The odds ratios (ORs) represent the odds for increasing from low to high viral load. RESULTS: Finally, 265 patients were grouped as normal (n = 125), CIN 1 (n = 51), CIN 2 (n = 23), CIN 3 (n = 46), and cervical cancer (n = 20). Among them, 139 (52.5%) had a low viral load, 90 (34.0) had a moderate viral load, and 36 (13.4%) had a high viral load. Taking the normal control group as a reference, a high viral load was an independent factor for CIN 1 (OR = 3.568, 95% CI: 1.164-10.941, P = 0.026), CIN 2 (OR = 6.939, 95% CI: 1.793-26.852, P = 0.005), CIN 3 (OR = 7.052, 95% CI: 2.304-21.586, P = 0.001), and cervical cancer (OR = 8.266, 95% CI: 2.120-32.233, P = 0.002). CONCLUSIONS: Among women who underwent cervical biopsy, higher high-risk HPV viral load in cervical lesions was associated with a higher risk of high-grade cervical lesions.

Hepatoprotective effect of forsythiaside a against acetaminophen-induced liver injury in zebrafish: Coupling network pharmacology with biochemical pharmacology.

ETHNOPHARMACOLOGICAL RELEVANCE: Forsythiae Fructus, the dried fruit of Forsythia suspensa (Thunb.) Vahl, is a commonly used traditional Chinese medicine and possesses various pharmacological activities, including anti-inflammation, anti-oxidant and liver protection. AIM OF THE STUDY: Although acetaminophen (APAP) has been frequently used for its antipyretic and analgesic effects, it leads to liver injury at an overdose or long-term medication. Forsythiaside A (FA), the principal active component of Forsythiae Fructus, exerts prominent antioxidant, anti-inflammatory and hepatoprotective effects. However, the protective property and underlying mechanism of FA against APAP challenge have not yet been elucidated. Therefore, we aimed to explore the hepatoprotective effect and action mechanism of FA against APAP-induced liver injury in zebrafish. MATERIALS AND METHODS: In this study, liver-specific transgenic zebrafish larvae (lfabp: EGFP) were used to investigate the protective effect of FA against overdose APAP exposure. The liver phenotype, morphological and biochemical assessments were carried out to evaluate the hepatoprotective effect of FA. Network pharmacology and molecular docking study were conducted to analyze the potential targets of FA in the treatment of APAP-induced liver injury. Finally, the mechanism of action was verified by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). RESULTS: The liver phenotype, morphological and biochemical assessments indicated that FA could mitigate APAP-triggered liver injury. Network pharmacology and molecular docking analysis indicated that the protective effect of FA might be related to the regulation of targets tumor necrosis factor (TNF), matrix metallopeptidase 9 (MMP9), matrix metallopeptidase 2 (MMP2), and phosphatidylinositol 3-kinase (PI3K). PCR results confirmed that FA could reverse the progressive alterations of genes involving in extracellular matrix remolding and PI3K/AKT-mediated apoptosis signaling pathway. CONCLUSIONS: Our results indicated that FA could mitigate APAP-induced liver injury through modulating the remolding of extracellular matrix and PI3K/AKT-mediated apoptosis.

Junctional and somatic hypermutation-induced CX4C motif is critical for the recognition of a highly conserved epitope on HCV E2 by a human broadly neutralizing antibody.

Induction of broadly neutralizing monoclonal antibodies (bNAbs) that bind to the viral envelope glycoproteins is a major goal of hepatitis C virus (HCV) vaccine research. The study of bNAbs arising in natural infection is essential in this endeavor. We generated a human antibody, 8D6, recognizing the E2 protein of HCV isolated from a chronic hepatitis C patient. This antibody shows broadly neutralizing activity, which covers a pan-genotypic panel of cell culture-derived HCV virions (HCVcc). Functional and epitope analyses demonstrated that 8D6 can block the interaction between E2 and CD81 by targeting a highly conserved epitope on E2. We describe how the 8D6 lineage evolved via somatic hypermutation to achieve broad neutralization. We found that the V(D)J recombination-generated junctional and somatic hypermutation-induced disulfide bridge (C-C) motif in the CDRH3 is critical for the broad neutralization and binding activity of 8D6. This motif is conserved among a series of broadly neutralizing HCV antibodies, indicating a common binding model. Next, the 8D6 inferred germline (iGL) was reconstructed and tested for its binding affinity and neutralization activity. Interestingly, 8D6 iGL-mediated relatively strong inhibition of the 1b genotype PR79L9 strain, suggesting that PR79L9 may serve as a potential natural viral strain that provides E2 sequences that induce bNAbs. Overall, our detailed epitope mapping and genetic studies of the HCV E2-specific mAb 8D6 have allowed for further refinement of antigenic sites on E2 and reveal a new mechanism to generate a functional CDRH3, while its iGL can serve as a probe to identify potential HCV vaccine strains.

HPV infection screening using surface plasmon resonance in women from Kunming (Southwest China).

No study examined the frequency of human papillomavirus (HPV) genotypes by surface plasmon resonance (SPR) in Southwest China. This was a cross-sectional survey (The Second Affiliated Hospital of Kunming Medical University, 10/2010 to 12/2011) in 150 patients who were hospitalized or volunteered for cervical cancer (CC) screening. A HPV typing kit was used to detect 24 types of HPV by the SPR technique. The HPV-positive rate was 34.8% in women with normal cytology and 92.9% in women with CC. The frequency of HPV16 increased from 9.4% for women with normal cytology to 28.9% for cervical intraepithelial neoplasia (CIN)1, 41.4% for CIN2, 54.1% for CIN3, and 71.4% for CC (p < 0.001). The frequency of HPV18 increased from 0% for women with normal cytology to 2.6% for CIN1, 3.4% for CIN2, 5.4% for CIN3, and 21.4% for CC (p = 0.03). HPV40 was only found in one patient with CC (p = 0.04). There was no relation between HPV genotype and women's age. In Kunming (Southwest China), the frequency of HPV infection was 74.0% among women who underwent CC screening. HPV16 and HPV18 were the two most frequent genotypes. SPR could be of value for the screening of HPV infection.

Human papillomavirus and human telomerase RNA component gene in cervical cancer progression.

This study aimed to examine hTERC gene in different grades of cervical intraepithelial neoplasia (CIN) and cervical cancer, and the association between hTERC and high risk-human papillomavirus (HR-HPV) infection. Patients who underwent cervical cancer screening at the Second Affiliated Hospital of Kunming Medical University between October 2010 and December 2011 were enrolled. All patients underwent liquid-based cytology test and hybrid capture 2 (HC2) for HPV detection. hTERC was examined using fluorescence in situ hybridization (FISH). Cervical colposcopy biopsy was performed if any of the three results was positive. HC2, FISH, and pathology were compared. A total of 1200 women underwent screening, 150 patients underwent cervical biopsy: 32 in the normal group, 38 in the CIN1 group, 66 in the CIN2/3 group, and 14 in the invasive cervical cancer group. More patients had HR-HPV infection in the CIN2/3 group and ICC group compared with the CIN1 group. hTERC increased with increasing histological dysplasia. There was significant difference in hTERC positive rate between each of the three groups. More patients with hTERC gene amplification were observed in the positive HR-HPV group than in the HR-HPV negative group. In conclusion, hTERC is a potential marker for precancerous cervical cancer lesions. hTERC might be correlated with HR-HPV infection in cervical diseases.

MicroRNA-214 Suppresses Ovarian Cancer by Targeting ß-Catenin.

BACKGROUND/AIMS: Ovarian cancer is one of the most common malignancies with a high rate of mortality in women. However, current therapies for ovarian cancer treatment are ineffective. Therefore, novel target identification is an urgent requisite. The present study aimed to investigate the role of microRNA-214 (miR-214) in ovarian cancer. METHODS: The expression of miR-214, ß-catenin, cyclin D1, c-myc, and TCF-1 at the transcriptional level was measured by real-time PCR, while that of ß-catenin, Cyclin D1, and c-Myc at the protein level were detected by western blot. Colony formation assay and transwell assay were used to explore the invasion ability of the cancer cells. Cell cycle was measured by flow cytometry. RESULTS: Real-time PCR showed that miR-214 expression in ovarian cancer cell lines was lower than that in the human normal ovarian epithelial cells, IOSE80. Furthermore, the low expression of miR-214 was correlated with high pathological grade. The rate of colony formation and invasion of miR-214 overexpression in SKOV-3 cells were weaker than that in control cells. Moreover, miR-214 overexpression led to the G0/G1 phase arrest. The expression of ß-catenin, Cyclin D1, and c-Myc was suppressed by the overexpression of miR-214. CONCLUSION: These results suggested that miR-214 may serve as a tumor suppressor of ovarian cancer by targeting the ß-catenin pathway.

Proportion of Uterine Malignant Tumors in Patients with Laparoscopic Myomectomy: A National Multicenter Study in China.

BACKGROUND: The Food and Drug Administration recently announced that the use of morcellation may cause fibroids or pelvic dissemination and metastasis of uterine sarcoma; therefore, the use of morcellation is limited in the USA. A large sample study is necessary to assess the proportion of uterine malignant tumors found in patients with laparoscopic myomectomy. METHODS: A national multicenter study was performed in China. From 2002 to 2014, 33,723 cases were retrospectively selected. We calculated the prevalence and recorded the clinical characteristics of the patients with malignancy after morcellation application. A total of 62 cases were finally pathologically confirmed as malignant postoperatively. Additionally, the medical records of the 62 patients were analyzed in details. RESULTS: The proportion of postoperative malignancy after morcellation application was 0.18% (62/33,723) for patients who underwent laparoscopic myomectomy. Nearly 62.9% (39/62) of patients had demonstrated blood flow signals in the uterine fibroids before surgery. And, 23 (37.1%) patients showed rapid growth at the final preoperative ultrasound. With respect to the pathological types, 38 (61.3%) patients had detectable endometrial stromal sarcoma, 13 (21.0%) had detectable uterine leiomyosarcoma, only 3 (3.2%) had detectable carcinosarcoma, and 5 (8.1%) patients with leiomyoma had an undetermined malignant potential. CONCLUSIONS: The proportion of malignancy is low after using morcellation in patients who undergo laparoscopic myomectomy. Patients with fast-growing uterine fibroids and abnormal ultrasonic tumor blood flow should be considered for malignant potential, and morcellation should be avoided.

MiR-214 suppressed ovarian cancer and negatively regulated semaphorin 4D.

Ovarian cancer is one of the most common human malignancies in women. MiR-214 and semaphorin 4D (sema 4D) were found to be abhorrently expressed and involved in the progress of several kinds of malignant cancers. This study is aimed to investigate the cellular role of miR-214 and demonstrate that miR-214 negatively regulated sema 4D in ovarian cancer cells. The data showed that miR-214 expression was consistently lower in ovarian cancer tissues and cells than those in the normal controls. Over-expression of miR-214 in ovarian cancer SKOV-3 cells inhibited cell proliferation and induced apoptosis. It was suggested that miR-214 functioned as the tumor suppressor in ovarian cancer. Bioinformatic analysis indicated that miR-214 possibly regulated sema 4D by binding the sema 4D messenger RNA (mRNA) 3'-untranslated region (UTR). Sema 4D mRNA and protein levels were up-regulated in ovarian cancer tissues and SKOV-3 cells. Up-regulation of miR-214 in SKOV-3 cell line suppressed the sema 4D expression in both protein and nucleic acid levels. While, down-regulation of miR-214 in SKOV-3 cells would increase sema 4D protein and nucleic acid expression levels. The effects of miR-214 up- and down-regulation on luciferase activities of wild-type (WT) sema 4D 3'-UTR were completely removed upon introduction of mutation in 3'-UTR of WT sema 4D. Therefore, the data also demonstrated that sema 4D was the direct target of miR-214 and was negatively regulated by miR-214 in ovarian cancer cells.

Correlation Between SNPs in Candidate Genes and VerifyNow-Detected Platelet Responsiveness to Aspirin and Clopidogrel Treatment.

PURPOSE: Patients with high on-treatment platelet reactivity (HPR) against aspirin or clopidogrel are at increased risk for adverse cardiovascular events. In this study, we explored the predictive value of common SNPs for the on-treatment platelet reactivity (OPR) against aspirin and clopidogrel assessed by VerifyNow assays. METHODS: This study recruited 286 Han Chinese individuals undergoing antiplatelet treatment, including 159 cases with aspirin only (100 mg/day) and 127 cases with dual therapy (aspirin 100 mg/day plus clopidogrel 75 mg/day) for at least 2 weeks. The OPR against aspirin and clopidogrel were assessed by VerifyNow Aspirin (ARU) and P2Y12 assays (PRU), respectively. Genotyping for the selected 25 SNPs within 11 genes and 2 GWAS loci was carried out by ABI multiplex SNaPshot method. RESULTS: The results indicated that rs4244285 (CYP2C19) and rs342293 (7q22.3) were significantly associated with PRU value (both P < 0.01). As for the OPR to aspirin, a weak statistical significance was observed in rs5445 (GNB3) (P = 0.049) and rs5758 (TBXA2R) (P = 0.045). After adjusting for the covariates including gender, age and smoking, carriers of allele A of rs4244285 remained as a strong predictor for HPR against clopidogrel. CONCLUSION: The current study suggests that common SNPs may predict OPR against clopidogrel as assessed by VerifyNow P2Y12, but are less likely to respond against aspirin as assessed by VerifyNow Aspirin.

[Autophagy and apoptosis of HeLa cells induced by recombinant human endostatin combined with hypoxia].

OBJECTIVE: To observe the impact of recombinant human endostatin (rhES) combined with hypoxia on the autophagy and apoptosis of human cervical cancer HeLa cells. METHODS: Under hypoxia, HeLa cells in logarithmic growth phase were treated with PBS, rhES, rhES combined with 3-methyladenine (3-MA, inhibitor of autophagy), respectively. Twenty-four hours later, cell apoptosis rate was detected using Hochest33528 staining and annexin V-FITC/PI staining and flow cytometry; the expressions of microtubule-associated protein light chain 3 (LC-3), Bcl-2, Bax proteins were determined using Western blotting. RESULTS: Hochest33528 staining suggested that cells of the hypoxia plus rhES group showed nuclear fragmentation; Annexin V-FITC/PI staining combined with flow cytometry revealed that the apoptosis rates were respectively (2.94±0.45)%, (21.38±0.92)% and (6.87±0.58)% in the three groups. It was significantly higher in the hypoxia plus rhES group than the other two groups. Western blotting showed that the expression levels of LC3 (LC-3II) and Bax increased and Bcl-2 decreased in the hypoxia plus rhES group as compared with the other two groups. CONCLUSION: Endostatin combined hypoxia can induce autophagy and apoptosis of HeLa cells.

[Clinical application of VerifyNow-P2Y12 assay in evaluation of platelet inhibition efficacy of clopidogrel].

OBJECTIVE: To evaluate the platelet inhibition efficacy in patients under regular maintenance dose of clopidogrel by VerifyNow-P2Y12 assay and explore the clinical characteristics of clopidogrel non-responders and related predicting factors. METHODS: A total of 99 patients underwent percutaneous coronary intervention procedure and receiving clopidogrel in regular maintenance dose for at least 1 week were enrolled. Platelet reactivity, including baseline, P2Y12 reaction unit (PRU), and platelet inhibition rate were measured with VeifyNow-P2Y12 assay. The dosage of anti-platelet drugs, combination with any other drugs, clinical characters in baseline of all enrolled patients were analyzed. PRU ≤ 240 was used as cut-off to identify clopidogrel responder and clopidogrel non-responder. In the non-responder group, patients were further separated into 3 sub-groups (types) according to the baseline and platelet inhibition rate: type I with high baseline, high inhibition rate, representing false non-responder; type II with low inhibition rate, representing true non-responder and type III mixed type. RESULTS: In this study, 48 of 99 patients were found to be clopidogrel non-responder (48.5%). The ratio of type I, type II and type III in the non-responder group was 9.1% (n = 9), 27.3% (n = 27), and 12.1% (n = 12), respectively. Baseline platelet value in female patients was significantly higher than in males (P < 0.01), number of females with high PRU also is higher than males (P < 0.01), female gender was a predict factor for type I non-responder (OR = 6.5, 95%CI 2.295 - 18.407, P < 0.01). BMI > 24 kg/m(2) was a risk factor for clopidogrel non-responder (P < 0.05), and may be regarded as a predict factor for type II non-responder (OR = 3.207, 95%CI 1.375 - 7.485, P < 0.01). Age, hypertension, diabetics, smoking, hyperlipidemia, CRP and pantoprazole use do not show significant correlation with baseline and platelet inhibition rate. CONCLUSIONS: Clopidogrel responses could be reliably detected by VerifyNow-P2Y12 assay. Female gender and high body weight are independent risk factors for clopidogrel non-responses.

Reciprocal regulation of Akt and Oct4 promotes the self-renewal and survival of embryonal carcinoma cells.

Signaling via the Akt serine/threonine protein kinase plays critical roles in the self-renewal of embryonic stem cells and their malignant counterpart, embryonal carcinoma cells (ECCs). Here we show that in ECCs, Akt phosphorylated the master pluripotency factor Oct4 at threonine 235, and that the levels of phosphorylated Oct4 in ECCs correlated with resistance to apoptosis and tumorigenic potential. Phosphorylation of Oct4 increased its stability and facilitated its nuclear localization and its interaction with Sox2, which promoted the transcription of the core stemness genes POU5F1 and NANOG. Furthermore, in ECCs, unphosphorylated Oct4 bound to the AKT1 promoter and repressed its transcription. Phosphorylation of Oct4 by Akt resulted in dissociation of Oct4 from the AKT1 promoter, which activated AKT1 transcription and promoted cell survival. Therefore, a site-specific, posttranslational modification of the Oct4 protein orchestrates the regulation of its stability, subcellular localization, and transcriptional activities, which collectively promotes the survival and tumorigenicity of ECCs.