[The miR-184 level in the seminal plasma exosome of male infertility patients and its clinical significance].

OBJECTIVE: To study the miR-184 level in the seminal plasma exosome of male infertility patients and its clinical significance. METHODS: Between 2015 and 2019, we collected 285 seminal plasma samples from 97 azoospermia (AS) and 96 asthenospermia (AZS) patients and 92 age-matched normal fertile controls in Jiangsu Provincial Hospital of Traditional Chinese Medicine, General Hospital of Eastern Theater Command and the First Hospital Affiliated to Wenzhou Medical University, identified the isolated seminal plasma exosomes by nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM) and Western blot, and detected the miR-184 level in the seminal plasma exosomes by quantitative real-time PCR (qRT-PCR). We determined the clinical value of the miR-184 level and its correlation with semen parameters by multiple statistics, predicted the target genes and involved pathways of miR-184 by bioinformatic algorithms, and analyzed their relationship with male infertility. RESULTS: NTA, TEM and Western blot exhibited plenty of exosomes in the seminal plasma of the patients. The results of qRT-PCR showed that the miR-184 level in the seminal plasma exosome was dramatically decreased in the AS patients compared with that in the normal fertile controls (0.227 ［0.092, 0.790］ vs 0.650 ［0.408, 1.061］, P < 0.01), but increased in AZS males in comparison with that in the control group (1.176 ［0.661, 1.946］ vs 0.650 ［0.408, 1.061］, P < 0.01). The areas under the ROC curve (AUC) for differentiating the AS and AZS patients from the controls were 0.866 (95% CI: 0.815－0.916) and 0.724 (95% CI: 0.653－0.795), respectively, and that for differentiating the AS from the AZS group was 0.964 (95% CI: 0.943－0.985). The miR-184 level in the seminal plasma exosome of the AZS patients was correlated positively with the sperm count (r = 0.243, P = 0.017) but negatively with the percentage of progressively motile sperm (r = －0.407, P = 0.006). Bioinformatics analysis indicated that the downstream target genes of miR-184 were significantly enriched in the protein regulatory pathways closely related to male reproduction and spermatogenesis. CONCLUSIONS: The miR-184 level in the seminal plasma exosome of infertility patients is significantly different from that of normal fertile males, which may serve as a potential auxiliary marker for the diagnosis of and participate in the development and progression of male infertility.

JQ1-Loaded Polydopamine Nanoplatform Inhibits c-MYC/Programmed Cell Death Ligand 1 to Enhance Photothermal Therapy for Triple-Negative Breast Cancer.

Programmed cell death ligand 1 (PD-L1) blockade has achieved great success in cancer immunotherapy; however, the response of triple-negative breast cancer (TNBC) to PD-L1 antibodies is limited. To address this challenge, we use the bromodomain and extra-terminal inhibitor JQ1 to down-regulate the expression of PD-L1 and thus elicit the immune response to TNBC instead of using antibodies to block PD-L1. JQ1 also inhibits the growth of TNBC as a targeted therapeutic agent by inhibiting the BRD4-c-MYC axis. The polydopamine nanoparticles (PDMNs) are introduced as a biodegradable and adaptable platform to load JQ1 and induce photothermal therapy (PTT) as another synergistic therapeutic modality. Because the JQ1-loaded PDMNs (PDMN-JQ1) are self-degradable and release JQ1 continuously, this synergistic treatment can lead to remarkable activation of cytotoxic T lymphocytes and induce a strong immune-memory effect to protect mice from tumor re-challenge. Taken together, our study demonstrates a compact and simple nanoplatform for triple therapy, including targeted therapy, PTT, and immunotherapy, for TNBC treatment.

[Difference of alkaloid components between old stems and tender stems of Gelsemium elegans].

This study aimed to comprehensively assess the difference of alkaloid components between old stems and tender stems of Gelsemium elegans by using ultra high-performance liquid chromatography coupled with photo-diode array and quadrupole time-of-flight mass spectrometry( UPLC-Q-TOF/MS~E) and high-performance liquid chromatography coupled with UV detector( HPLC-UV). Firstly,the different components in old stems and tender stems were analyzed by UHPLC-Q-TOF/MSEcombined with principal component analysis( PCA) and orthogonal partial least squares discriminant analysis( OPLS-DA),respectively. As a result,17 major different components were found. At the same time,the distribution of these alkaloids in old stems and tender stems was determined,and the alkaloids with higher polarity are relatively higher in the tender stems,while the old stems are in the opposite case. In addition,three main components in the G. elegans were quantified by HPLC-UV. The results showed that the contents of koumine and humantenmine in old stems were higher than those in tender stems,and the content of gelsemine in tender stems was relatively high. This study systematically evaluated the differences of alkaloids between the old stems and tender stems of G. elegans,and quantified the main three alkaloids. It laid the foundation of the safe and effective application of G. elegans.

The qualitative and quantitative analyses of Gelsemium elegans.

Gelsemium elegans is a traditional Chinese medicine that has been used to treat eczema, bruises, rheumatoid arthritis and skin ulcers for many years, and alkaloids are its major active and toxic constituents. This study aimed to comprehensively assess the quality of G. elegans samples including different plant parts and origins using ultra high-performance liquid chromatography coupled with photo-diode array and quadrupole time-of-flight mass spectrometry (UHPLC-PDA-QTOF/MS) and high-performance liquid chromatography coupled with UV detector (HPLC-UV). Firstly, the UHPLC-PDA-QTOF/MS approach was developed for the characterization of alkaloids in G. elegans and understanding the differences between multiple groups of samples. Based on the exact mass information, the fragmentation characteristics and the retention time of compounds, 38 alkaloids were identified or tentatively identified. 24 potential chemical markers for differentiating different plant parts of G. elegans were selected through PCA and OPLS/PLS-DA analysis. Secondly, a heatmap visualization was employed for clarifying the distribution of 24 selected alkaloids with high response in the UV. The roots, stems and leaves from Yunnan Province possess relatively consistent alkaloids composition, respectively. Most compounds in the root have a higher content than stems and leaves. Thirdly, a HPLC-UV approach was developed for quantitative analysis of three major alkaloids (gelsemine, koumine and gelsenicine) of G. elegans, and the results showed remarkable variation in the contents of these constituents. While, the contents of three alkaloids fluctuate relatively less in the stem. These results indicated that integrated chemical profiling and quantitative analysis of alkaloids in G. elegans from different plant parts and origins could be assessed by this method, which would establish the foundation for the application of G. elegans.

Expression of AQP2, AQP4 and AQP 8 in mouse intestine induced by unprocessed and processed Euphorbia lathyris.

The present research was designed to study expression of AQP2, AQP4 and AQP8 in mouse intestines induced by unprocessed and processed Euphorbia lathyris. KM mice were given by different dose lavage of unprocessed and processed Euphorbia lathyris, Euphorbia factor L1, Euphorbia factor L2, Euphorbia factor L3. Samples of mouse intestine were collected for protein levels of AQP2, AQP 4 and AQP 8 which were assessed by immunohistochemical staining and mRNA expression of AQP2, AQP 4 and AQP 8 which were quantified by Real Time-PCR. Comparing to the normal control group, the protein levels of AQP2, AQP 4 and AQP 8 were significantly decreased (P<0.05)by Semen Euphorbiae group and Semen Euphorbiae Pulveratum group (unprocessed and processed Euphorbia lathyris) induced. Protein expression of AQP2, AQP 4 and AQP 8 in the Euphorbia factor L1, Euphorbia factor L2 and Euphorbia factor L3 group were not significantly lower than normal control group. There had no differences on the levels of AQP2 and AQP 8 mRNA expressions between the high-dose group of semen Euphorbiae group, semen Euphorbiae Pulveratum group and positive control group, while significantly lower than normal control group (P<0.05). Expression of AQP4 mRNA in the Semen Euphorbiae group and Semen Euphorbiae Pulveratum group has not significantly decreased. But levels of AQP2, AQP 4 and AQP 8 mRNA in the Euphorbia factor L1 group had no significant differences in normal control group and positive control group. These findings suggest that semen Euphorbiae could regulate expression of AQP2, AQP 4 and AQP 8 protein and mRNA, which may be the possible one reason of semen Euphorbiae induces diarrhea. The semen Euphorbiae group has more significant effects on the levels of AQP2, AQP 4 and AQP 8 protein and mRNA than semen Euphorbiae Pulveratum group, which may be one of the mechanisms of processing attenuation.

Photosensitizer-loaded biomimetic platform for multimodal imaging-guided synergistic phototherapy.

Photodynamic therapy (PDT) has attracted much attention as a strategy for tumor therapy. However, the insolubility and poor tumor-targeting ability of most photosensitizers (PSs) hinder PDT from further development. Therefore, it is necessary to explore new carriers with good water solubility and biocompatibility to deliver PSs to tumors. Melanin nanoparticles are novel biomimetic nanocarriers with excellent biocompatibility, loading capacity, photothermal therapy (PTT) and magnetic resonance (MR)/photoacoustic (PA) imaging properties. Here we designed polydopamine melanin nanoparticles (PDMNs) as a delivery platform for the photosensitizer Chlorin e6 (PDMN-Ce6) and realized its application as a theranostic agent for tumor therapy. The PDMN-Ce6 exhibited excellent biocompatibility, good water solubility and high loading capability (35.2 wt%) for Ce6. Compared with the free Ce6, PDMN-Ce6 showed higher cellular internalization and superior synergistic phototherapy effects in an in vitro study. An in vivo study indicated that the accumulation of PDMN-Ce6 at tumor sites was 2.8-fold higher than that of free Ce6 at 24 h post-injection, which was beneficial for MR/PA imaging. Moreover, the synergetic therapy significantly inhibited tumor growth, causing tumor necrosis and tumor angiogenesis suppression. These results suggest that our biomimetic and biocompatible platform could improve the delivery of Ce6 to tumors and realize multimodal imaging-guided tumor synergetic phototherapy.

[Chromatographic fingerprint analysis of Toosendan Fructus by HPLC-CAD coupled with chemometrics methods].

A new method was developed for the chromatographic fingerprint analysis of Toosendan Fructus by HPLC coupled with the charged aerosol detector (CAD) in the present study. Samples were well separated on an Agilent ZOBAX SB C18 column (4.6 mm × 250 mm, 5 µm) by gradient elution using acetonitrile and water containing 0.1 % formic acid (v/v) at the flow rate of 1.0 mL·min−1. The column temperature was 30 ℃ and the injection volume was 5 µL. The nitrogen inlet pressure of the charged aerosol detector (CAD) was 35 psi, and the nebulizer chamber temperature was 35 ℃. In addition, the method of the chromatographic fingerprints combined with multivariate statistical analysis was effective and reasonable lead to an accurate classification of 20 batches of samples from different locations. The results showed that 28 common peaks were observed in the fingerprint and the samples were classified into three clusters. The established method was well validated, and showed high precision, good repeatability, and satisfactory stability. It may serve in the quality control and evaluation of Toosendan Fructus.

Studies of the microbial metabolism of flavonoids extracted from the leaves of Diospyros kaki by intestinal bacteria.

Flavonoid glycosides are metabolized by intestinal bacteria, giving rise to a wide range of phenolic acids that may exert systemic effects in the body. The microbial metabolism of flavonoids extracted from the leaves of Diospyros kaki (FLDK) by intestinal bacteria was investigated in vitro. High-performance liquid chromatography/linear trap quadrupole orbitrap mass spectrometry was performed to analyze the metabolites of flavonoids in vivo using Xcalibur2.1 software. The results showed that the levels of flavonoid glycosides and flavonoid aglycones decreased rapidly in the process of microbial metabolism by intestinal bacteria in vitro, and the metabolic rate may be related to the concentration of intestinal bacteria in the culture solution. In vivo metabolites of FLDK were detected in rat plasma and urine after oral administration of FLDK. Eight flavonoids were identified in the urine, and three were identified in the plasma; however, flavonoid aglycones were not found in the plasma.