Ethyl acetate extract of Gastrodia elata protects Caenorhabditis elegans from oxidative stress and amyloid ß peptide toxicity.

Gastrodia elata Blume is a traditional Chinese medicine with a long history, which has numerous pharmacological activities, such as anti-inflammation, anti-oxidation and protection of nerves. The present study investigated the regulatory effect of ethyl acetate extract of Gastrodia elata (EEGE) on the ß-amyloid (Aß) toxicity of Caenorhabditis elegans (C. elegans). First, the main components of EEGE were analyzed using high-performance liquid chromatography, and the total phenols, total flavonoids and total antioxidant capacity of EEGE were determined. Next, the regulation effect of EEGE on Aß-induced toxicity of C. elegans was evaluated through experiments on nematode paralysis, lifespan, oxidative and heat stress, locomotor ability, reproductive ability, reactive oxygen species (ROS) level, Aß aggregation test, malondialdehyde (MDA) level, catalase (CAT) activity and superoxide dismutase (SOD) activity. Finally, the mechanism of EEGE was elucidated using RNA sequencing (RNA-Seq) and the expression levels of related genes were verified using quantitative PCR. The present study revealed that the main components of EEGE included phosphorylated (p)-hydroxybenzyl alcohol, p-hydroxybenzaldehyde and 4,4'-dihydroxydiphenylmethane, possessing strong in vitro free radical scavenging and reducing abilities. In addition, after the intervention of EEGE, the paralysis of nematodes could be delayed, the survival time of the nematodes was prolonged, the survival rate of the nematodes under stress (high temperature and oxidation) conditions was improved, the activity capacity and reproductive capacity of the nematodes were improved, the activities of SOD and CAT were improved and the levels of ROS and MDA were reduced. Notably, EEGE directly inhibited Aß plaque aggregation in nematodes. RNA-Seq analysis showed that EEGE regulated metabolism and longevity-related genes, and these genes were regulated by the insulin/IGF-1 signaling (IIS) pathway. Therefore, the present study hypothesized that the regulatory mechanism of EEGE was significantly related to the IIS pathway. The present research results demonstrated that the protective effect of EEGE on transgenic C. elegans was to reduce Aß protein aggregation, improve the in vivo antioxidant level, effectively remove free radicals and to regulate the expression of genes related to IIS pathway, thereby reducing Aß-induced toxicity and delaying nematode paralysis.

Protective effect of Gastrodia elata Blume in a Caenorhabditis elegans model of Alzheimer's disease based on network pharmacology.

The aim of the present study was to investigate the protective effect of Gastrodia elata Blume (GEB) against Caenorhabditis elegans (C. elegans) in Alzheimer's disease (AD) through network pharmacology. Firstly, the active constituents of GEB through ETCM and BATMAN-TCM databases were collected and its potential AD-related targets in Swiss Target Prediction were predicted. The potential targets related to AD were collected from the GeneCards, OMIM, CTD and DisGeNET databases, and the differential genes (DEGs) between the normal population and the AD patient population in GSE5281 chip of the Gene Expression Omnibus database were collected at the same time. The intersection of the three targets yielded 59 key targets of GEB for the treatment of AD. The drug-active ingredient-target-AD network diagram was constructed and visualized with Cytoscape software to obtain the core components. Subsequently, protein-protein interaction analysis (PPI) was performed on 59 key targets through STRING database, and Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses was performed on 59 key targets. Finally, molecular docking was conducted between core components and core targets using AutoDock software, and the C. elegans AD model was used for experimental verification to explore the regulatory paralysis effect of core components on the C. elegans model, ß-amyloid (Aß) plaque deposition, and quantitative polymerase chain reaction verification of the regulatory effect of components on targets. The GEB components 4,4'-dihydroxydiphenyl methane (DM) and protocatechuic aldehyde (PA) were found to be most strongly associated with AD, and five core targets were identified in the PPI network, including GAPDH, EP300, HSP90AB1, KDM6B, and CREBBP. In addition to GAPDH, the other four targets were successfully docked with DM and PA using AutoDock software. Compared with the control group, 0.5 mM DM and 0.25 mM PA significantly delayed C. elegans paralysis (P<0.01), and inhibited the aggregation of Aß plaques in C. elegans. Both DM and PA could upregulate the expression level of core target gene HSP90AB1 (P<0.01), and DM upregulated the expression of KDM6B (P<0.01), suggesting that DM and PA may be potential active components of GEB in the treatment of AD.

Identification and Verification of Ubiquitin D as a Gene Associated with Hepatitis C Virus-Induced Hepatocellular Carcinoma.

INTRODUCTION: Accumulated studies have suggested that hepatitis C virus (HCV) infection is one of the leading causes for hepatocellular carcinoma (HCC). However, the mechanisms underlying the effect of HCV on the occurrence of HCC are still poorly understood. METHODS: HCV infection datasets (GSE82177 and GSE17856) and HCC datasets (The Cancer Genome Atlas Liver Hepatocellular Carcinoma and GSE89377) were downloaded from Gene Expression Omnibus or TCGA for analysis. The common differentially expressed genes in the above four datasets were identified by R software. The expression of ubiquitin D (UBD) in HCV-infected HepG2 cells was detected by RT-qPCR and Western blot, respectively. The interaction between NS3 and p53 was detected by co-immunoprecipitation. The influence of UBD on the proliferation and migration ability of HepG2 cells was evaluated by CCK-8 and wound healing assay, respectively. RESULTS: UBD was upregulated in both HCV-infected samples and HCC samples. HCV NS3 interacted with p53 and inhibited its expression. HCV NS3-induced UBD promoted the proliferation and migration of HepG2 cells. CONCLUSION: Our results suggest that HCV NS3-induced UBD is positively correlated with the development of HCV-related HCC during HCV infection. Targeting UBD could be a potential strategy for preventing and treating HCV-induced HCC.

Influence of miRNA-30a-5p on Pulmonary Fibrosis in Mice with Streptococcus pneumoniae Infection through Regulation of Autophagy by Beclin-1.

The study is aimed at observing the influence of microribonucleic acid- (miRNA-) 30a-50p on the pulmonary fibrosis in mice with Streptococcus pneumoniae infection through the regulation of autophagy by Beclin-1. Specific pathogen-free mice were instilled with Streptococcus pneumoniae through the trachea to establish the pulmonary fibrosis model. Then, they were divided into the miRNA-30a-50p mimics group (mimics group, n = 10) and miRNA-30a-5p inhibitors group (inhibitors group, n = 10), with the control group (n = 10) also set. Pulmonary tissue wet weight/dry weight (W/D) was detected. The content of tumor necrosis factor-α (TNF-α), interleukin- (IL-) 6, and myeloperoxidase (MPO) was determined using enzyme-linked immunosorbent assay (ELISA). Besides, the changes in the pulmonary function index dynamic lung compliance (Cdyn), plateau pressure (Pplat), and peak airway pressure (Ppeak) were monitored, and the gene and protein expression levels were measured via quantitative PCR (qPCR) and Western blotting. The expression level of miRNA-30a-5p was substantially raised in the mimics group (p < 0.05), but extremely low in the inhibitors group (p < 0.05). The mimics group had obviously raised levels of serum aminotransferase (AST), glutamic-pyruvic transaminase (GPT), alkaline phosphatase (ALP), and pulmonary tissue W/D (p < 0.05). Additionally, the expression levels of TNF-α, IL-6, and MPO were notably elevated in the mimics group, while their expression levels showed the opposite conditions in the inhibitors group (p < 0.05). According to the HE staining results, the inhibitors group had arranged orderly cells, while the mimics group exhibited lung injury, pulmonary edema, severe inflammatory response, and alveolar congestion. In the inhibitors group, Cdyn was remarkably elevated, but Pplat and Ppeak declined considerably (p < 0.05). Besides, the inhibitors group exhibited elevated messenger RNA (mRNA) levels of Beclin-1 and LC3, lowered mRNA levels of α-SMA and p62, a raised protein level of Beclin-1, and a markedly decreased protein level of p62 (p < 0.05). Silencing miRNA-30a-5p expression can promote the expression of Beclin-1 to accelerate the occurrence of autophagy, thereby treating pulmonary fibrosis in mice with Streptococcus pneumoniae infection.