Extracellular vesicle-derived miR-146a as a novel crosstalk mechanism for high-fat induced atherosclerosis by targeting SMAD4.

INTRODUCTION: Exosome-miR-146a is significantly increased in patients with Atherosclerosis (AS), but its mechanism and effect on AS have not been fully elucidated. OBJECTIVES: To explore the change rule and mechanism of exosomes release, and the role and molecular mechanism of exosome-miR-146a in AS. METHODS: We isolated and identified exosomes from THP-1 macrophages after treating them with ox-LDL. Then used co-immunoprecipitation and silver staining to identify the proteins involved in regulating exosome release. PKH67 was used to label exosomes to confirm that cells can absorb them, and then co-culture with HVSMCs for cell proliferation and migration detection. The target genes of miR-146a were screened and identified through bioinformatics and luciferase activity assay, and the expression of miR-146a and related proteins was detected through qRT-PCR and Western blot in HUVECs. An AS model in LDLR-/- mice induced by a high-fat diet was developed to investigate the impact of exosome-miR-146a on AS. RESULTS: The results showed that experimental foam cells from AS showed higher expression of miR-146a. It was observed that NMMHC IIA and HSP70 interacted to regulate the release of exosomes. And HUVECs can absorb exosomes derived from macrophages. In addition, we also found that miR-146a directly targeted the SMAD4 gene to modulate the p38 MAPK signaling pathway, thereby mediating HUVECs damage. Furthermore, exosome-miR-146a induced abnormal proliferation and migration of HVSMCs. The expression of miR-146a was significantly reduced in miR-146a-mimics mice and increased in miR-146a inhibitor mice whereas the inhibition of miR-146a effectively reduced while increasing miR-146a worsened AS in mice. CONCLUSION: Our findings expressed the potential of miR-146a as a favorable therapeutic target for AS, however, further exploration is suggestive for deep understanding of the mechanisms regulating exosome-miR-146a release in vivo and to develop effective therapeutic strategies involving miR-146a.

An anti-inflammatory active ingredients in Poriae cutis: Screening based on network pharmacology.

Hyperuricemia (HUA) is a disorder of uric acid metabolism, which can lead to the formation of gouty arthritis, kidney inflammation and other damages. Previous studies have found that the alcohol extract of Poria cutis can reduce the level of uric acid and protect against kidney injury. Based on network pharmacology, the core targets and main active components of P. cutis intervention in HUA were determined. Most of the potential active ingredients are triterpenoid acids such as tumulosic acid (TA) and eburicoic acid (EA), and the potential targets are TNF and IL-6, which are associated with inflammation. In vitro experiments have shown that TA can significantly inhibit the release of NO, TNF-α and IL-6 in inflammatory RAW264.7 cell culture medium and the expression of TNF-α and IL-6 in RAW264.7 cells. This study suggests that TA based on network pharmacological screening has obvious anti-inflammatory effect on inflammatory RAW264.7 cells and is a promising anti-inflammatory compound.

Poria cocos Attenuated DSS-Induced Ulcerative Colitis via NF-κB Signaling Pathway and Regulating Gut Microbiota.

Ulcerative colitis (UC), as a chronic inflammatory disease, presents a global public health threat. However, the mechanism of Poria cocos (PC) in treating UC remains unclear. Here, LC-MS/MS was carried out to identify the components of PC. The protective effect of PC against UC was evaluated by disease activity index (DAI), colon length and histological analysis in dextran sulfate sodium (DSS)-induced UC mice. ELISA, qPCR, and Western blot tests were conducted to assess the inflammatory state. Western blotting and immunohistochemistry techniques were employed to evaluate the expression of tight junction proteins. The sequencing of 16S rRNA was utilized for the analysis of gut microbiota regulation. The results showed that a total of fifty-two nutrients and active components were identified in PC. After treatment, PC significantly alleviated UC-associated symptoms including body weight loss, shortened colon, an increase in DAI score, histopathologic lesions. PC also reduced the levels of inflammatory cytokines TNF-α, IL-6, and IL-1ß, as evidenced by the suppressed NF-κB pathway, restored the tight junction proteins ZO-1 and Claudin-1 in the colon, and promoted the diversity and abundance of beneficial gut microbiota. Collectively, these findings suggest that PC ameliorates colitis symptoms through the reduction in NF-κB signaling activation to mitigate inflammatory damage, thus repairing the intestinal barrier, and regulating the gut microbiota.

Timosaponin BII inhibits TGF-ß mediated epithelial-mesenchymal transition through Smad-dependent pathway during pulmonary fibrosis.

Pulmonary fibrosis (PF) is a progressive and fatal interstitial lung disease with limited therapeutic options at present, and epithelial-mesenchymal transition (EMT) is recognized as a major cause of lung fibrosis. Our previous work has confirmed that total extract of Anemarrhena asphodeloides Bunge [Asparagaceae] exerted the effect of anti-PF. As a main constituent of Anemarrhena asphodeloides Bunge [Asparagaceae], the effect of timosaponin BII (TS BII) on drug-induced EMT process in PF animals and alveolar epithelial cells remains unknown. In this study, we evaluated the effect of TS BII on bleomycin (BLM)-induced PF. The results showed that TS BII could restore the structure of lung architecture and MMP-9/TIMP-1 balance in fibrotic rat lung and inhibit collagen deposition. Moreover, we found that TS BII could reverse the abnormal expression of TGF-ß1 and EMT-related marker proteins including E-cadherin, vimentin, and α-SMA. Besides, aberrant TGF-ß1 expression and phosphorylation of Smad2 and Smad3 in BLM-induced animal model and TGF-ß1-induced cell model were downregulated by TS BII treatment, indicating that EMT in fibrosis was suppressed by inhibition of TGF-ß/Smad pathway both in vivo and in vitro. In summary, our study suggested that TS BII could be a promising candidate for PF treatment.

The SAR and action mechanisms of autophagy inhibitors that eliminate drug resistance.

Autophagy is an essential homeostatic and catabolic process crucial for the degradation or recycling of proteins and cellular components. Drug resistance has been demonstrated to be closely implicated in increased autophagy. Autophagy inhibition to reverse drug resistance involves in the five stages of autophagy, including phagophore initiation, vesicle nucleation, vesicle elongation, vesicle fusion and cargo degradation. Herein, emphases were placed on discussions on the targets responsible for the upstream phagophore initiation and nucleation of autophagosome, as well as the ones mediating the downstream autophagosome and lysosome fusion and cargo degradation. The structure-activity relationships (SARs) and action mechanisms of the corresponding target-based small molecule autophagy inhibitors were analyzed and delineated. This review will provide a promising guidance for the design and optimization of drug-like scaffolds in the discovery of autophagy inhibitors able to eliminate drug resistance.

A Rapid and Efficient Strategy for Quality Control of Clinopodii herba Encompassing Optimized Ultrasound-Assisted Extraction Coupled with Sensitive Variable Wavelength Detection.

Clinopodii herba is a folk herbal medicine for treatments of hemorrhagic disorders. However, there is not even a quantitative standard for clinopodii herba deposited in the Chinese Pharmacopoeia. The development of a strategy for rapid and efficient extraction and simultaneous detection of multiple components in clinopodii herba is therefore of great value for its quality evaluation. Here, a variable wavelength strategy was firstly applied to quantity multiple components by segmental monitoring by UHPLC with diode array detector following ultrasound-assisted extraction. The parameters of ultrasound-assisted extraction were optimized using single factor optimization experiments and response surface methodology by a Box-Behnken design combined with overall desirability. Subsequently, a rapid, efficient, and sensitive method was applied for simultaneous determination of eleven compounds, which represented the major and main types of components in clinopodii herba. Moreover, the performance of the validated method was successfully applied for the quality control of various batches of clinopodii herba and provided sufficient supporting data for the optimum harvest time. The Box-Behnken-optimized ultrasound-assisted extraction coupled with variable wavelength detection strategy established in this work not only improves the quality control of clinopodii herba, but also serves as a powerful approach that can be extended to quality evaluation of other traditional Chinese medicines.

Total extract of Anemarrhenae Rhizoma attenuates bleomycin-induced pulmonary fibrosis in rats.

Pulmonary fibrosis is a progressive interstitial lung disease with poor prognosis. Anemarrhenae Rhizoma is a traditional Chinese herbal medicine and has been applied in clinical practice for a long history. Recently, components of Anemarrhenae Rhizoma were reported to possess anti-inflammatory and immunomodulatory features; however, the effect of them on pulmonary fibrosis remains unknown. In this study, we explored the therapeutic effect of total extract of Anemarrhenae Rhizoma (TEAR) on bleomycin-induced pulmonary fibrosis. Pulmonary fibrosis rat model was established by a single intratracheal instillation of bleomycin, three doses of TEAR were intragastrically administered for consecutive 28 days. Subsequent to sacrificing of rats, pulmonary fibrosis was observed in rats treated with bleomycin, but administration of TEAR attenuated lung fibrosis, as evidenced by the improved lung histopathological damage and decreased weight loss and lung index. Moreover, TEAR treatment inhibited the inflammatory response in lung fibrosis, which was shown by the reduced nitrogen oxide level and myeloperoxidase activity. Furthermore, TEAR modulated the redox balance in lung tissue by alleviated lipid peroxidation and enhanced enzymatic antioxidants activity. Meanwhile, TEAR protected the rats from fibrosis in a dose-dependent manner, and the anti-fibrotic activity of TEAR may be related to the modulation of TGF-ß1/Smad signaling pathway. Collectively, TEAR alleviates bleomycin-induced pulmonary fibrosis, indicating perspectives for development of a potential agent for lung fibrosis therapy.

Integration of full-length transcriptomics and targeted metabolomics to identify benzylisoquinoline alkaloid biosynthetic genes in Corydalis yanhusuo.

Corydalis yanhusuo W.T. Wang is a classic herb that is frequently used in traditional Chinese medicine and is efficacious in promoting blood circulation, enhancing energy, and relieving pain. Benzylisoquinoline alkaloids (BIAs) are the main bioactive ingredients in Corydalis yanhusuo. However, few studies have investigated the BIA biosynthetic pathway in C. yanhusuo, and the biosynthetic pathway of species-specific chemicals such as tetrahydropalmatine remains unclear. We performed full-length transcriptomic and metabolomic analyses to identify candidate genes that might be involved in BIA biosynthesis and identified a total of 101 full-length transcripts and 19 metabolites involved in the BIA biosynthetic pathway. Moreover, the contents of 19 representative BIAs in C. yanhusuo were quantified by classical targeted metabolomic approaches. Their accumulation in the tuber was consistent with the expression patterns of identified BIA biosynthetic genes in tubers and leaves, which reinforces the validity and reliability of the analyses. Full-length genes with similar expression or enrichment patterns were identified, and a complete BIA biosynthesis pathway in C. yanhusuo was constructed according to these findings. Phylogenetic analysis revealed a total of ten enzymes that may possess columbamine-O-methyltransferase activity, which is the final step for tetrahydropalmatine synthesis. Our results span the whole BIA biosynthetic pathway in C. yanhusuo. Our full-length transcriptomic data will enable further molecular cloning of enzymes and activity validation studies.

Gastrodin prevents homocysteine-induced human umbilical vein endothelial cells injury via PI3K/Akt/eNOS and Nrf2/ARE pathway.

In this study, we investigated the protective effects of gastrodin (Gas) against homocysteine-induced human umbilical vein endothelial cell (HUVEC) injury and the role of the phosphoinositide 3-kinase (PI3K)/threonine kinase 1 (Akt)/endothelial nitric oxide synthase (eNOS) and NF-E2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathways. We stimulated cells with homocysteine (1 mmol/L, 24 hours) and tested the effects of gastrodin (200-800 µg/mL) on cell viability and the production of malondialdehyde (MDA), lactate dehydrogenase (LDH) and reactive oxygen species (ROS). Then, Nrf2 distribution in the cytoplasm and nucleus as well as the expression of enzymes downstream of Nrf2 was determined. Furthermore, we analysed the expression of bax, bcl-2 and cleaved caspase3, and assessed the involvement of the PI3K/Akt/eNOS pathway by Western blots. Finally, we tested the vasoactive effect of gastrodin in thoracic aortic rings. The results showed that gastrodin decreased MDA, LDH and ROS production and increased cell viability, NO production and relaxation of thoracic aortic rings. Moreover, the protective effects of Gas on NO production and relaxation of thoracic aortic rings were blocked by L-NAME but enhanced by Cav-1 knockdown, and MK-2206 treatment abolished the effect of Gas on the ROS. In addition, treatment with gastrodin increased Nrf2 nuclear translocation, thus enhancing the expression of downstream enzymes. Finally, gastrodin increased the expression of PI3K, p-Akt, and eNOS and decreased Cav-1 protein expression. In conclusion, our study suggested that gastrodin may protect HUVECs from homocysteine-induced injury, and the PI3K/Akt/eNOS and Nrf2/ARE pathways may be responsible for the efficacy of gastrodin.

Molecular networking uncovers steroidal saponins of Paris tengchongensis.

Based on a method combining the LC-MS/MS molecular networking strategy with the conventional means of phytochemical research, the chemical constituents and the availability of Paris tengchongensis, a new species found in 2017 from Yunnan Province, were investigated for the first time. The molecular networking showed that this species contained the characteristic steroidal glycosides of the genus Paris by comparison of those of Paris polyphylla var. yunnanensis. Furthermore, the detailed investigation on the 80% EtOH extract of its rhizomes resulted to the isolation of twenty steroidal glycosides including three new spirostane-type saponins, named paristengosides A-C (1-3). Their structures were confirmed by spectroscopic analyses (HRMS and NMR) and chemical methods. The new isolates were evaluated for their cytotoxicities against two human cancer cell lines (HEL and MDA-MB-231), anti-inflammatory effects on a lipopolysaccharide (LPS)-stimulated NO production model in RAW264.7 macrophages, anti-AChE, and antimicrobial activities. The results from the molecular networking and the investigation on the chemical constituents suggested that P. tengchongensis can be used as a potential resource of Rhizoma Paridis.

Identification and Selection of Reference Genes for Quantitative Transcript Analysis in Corydalis yanhusuo.

Key word: qPCR; Corydalisyanhusuo; reference genes; geNorm; NormFinder; BestKeeper.

Comparison of α-glucosidase inhibitory effect and bioactive constituents of Anemarrhenae Rhizoma and Fibrous Roots.

Comprehensive utilization of medicinal plant resources is of great significance for sustainable development of traditional Chinese medicines. In the present study, the α-glucosidase inhibitory activities of the rhizome and fibrous root of Anemarrhena Asphodeloides Bunge, were compared detailedly, and a high performance liquid chromatography coupled with electrospray ionization tandem triple quadrupole mass spectrometry (HPLC-QQQ/MS) method was developed for simultaneous quantification of seven bioactive constituents including neomangiferin, mangiferin, isomangiferin, timosaponin BII, timosaponin B, timosaponin AIII, and timosaponin N in 40 batches of samples. The results demonstrated that fibrous root extracts had more potent α-glucosidase inhibitory activity than rhizome extracts. Mangiferin and isomangiferin were abundant in fibrous root, while the analyzed saponins were rich in rhizome. Based on the chemometrics methods including principal component analysis (PCA), orthogonal partial least square discriminant analysis (OPLS-DA), and partial least square (PLS), mangiferin and isomangiferin might be mainly responsible for α-glucosidase inhibitory activity of the genus. These findings indicate that the established HPLC-QQQ/MS method was proven to be useful and efficient for quality control of Anemarrhena materials, and fibrous root had the potential to be utilized as anti-diabetic medicinal resource.

Orthogonal separation protocol for the simultaneous preparation of four medically active compounds from Anemarrhenae Rhizoma by sequential polyamide and macroporous resin adsorbent chromatography.

The green and efficient preparation of natural products from biomass is considered an important area of interest in pharmaceutical applications. In this study, we aimed to provide a practical example with a popular traditional Chinese medicine, Anemarrhenae Rhizome, and showcase the orthogonal use of column chromatography with polyamide and macroporous adsorption resins to selectively concentrate and efficiently purify four bioactive compounds: neomangiferin (NMF), mangiferin (MF), timosaponin B-II (TS B-II), and timosaponin A-III (TS A-III). First, polyamide T60-100 was employed to fractionalize the crude extracts of Anemarrhenae Rhizome. Macroporous resin HPD400 was subsequently used to purify the xanthones and steroidal saponins. Under the optimized conditions, 2.31 g of NMF, 4.10 g of MF, 12.87 g of TS B-II, and 2.78 g of TS A-III were prepared from 1 kg of crude materials, and their purities were 90.0, 92.15, 90.8, and 92.61%, respectively. The results of this study indicate that the combined column chromatography with polyamide and macroporous adsorption resins can be referenced as a green and efficient alternative for large-scale purification of bioactive ingredients from herbal raw materials.

[The extraction technology of epigoitri from isatidis radix by supercritical CO2 fluid].

OBJECTIVE: To study the extraction technology of epigoitri from Isatidis Radix by supercritical CO2 fluid. METHODS: The effects of pressure, temperature, time, concentration and dosage of alcohol were studied by single factor analysis and orthogonal test. RESULTS: The optimized conditions were as follows: The pressure was 20 MPs, the temperature was 50 degrees C, the time was 2 h, concentration of alcohol was 100%, dosage was 80 mL. The content of epigoitri in the extract could reach 38.63% under the above conditions. CONCLUSION: This method is simple, rapid and it is suitable for the extraction of epigoitri from Isatidis Radix.