Identification of miRNAs in a Liver of a Human Fetus by a Modified Method

BACKGROUND: miRNAs are 17-25 nucleotides long RNA molecules that have been found to regulate gene expression in human cells. There are studies showing that different groups of miRNAs are involved in development of different tissues. In hepatocytes there are reported particular types of miRNAs that regulate gene expression. METHODS: We established a human fetal liver cDNA library by a modified cloning protocol. Then plasmid isolation from the colonies was performed. After sequencing and database searching, the miRNAs were recognized. RT-PCR and sequencing were carried out to validate the miRNAs detected. Real-time PCR was used to analyze the expression of each miRNA. RESULTS: One novel miRNA was discovered, together with another 35 previously-known miRNAs in the fetal liver. Some of them existed in variants. The miRNAs identified were validated by RT-PCR and sequencing. Quantitative analysis showed that they have variable expression. CONCLUSION: Our results indicate that a special group of miRNAs may play an important role in fetal liver development in a synergistic manner

Salidroside alleviates UVB-induced skin damage by inhibiting keratinocytes pyroptosis via the AQP3/ROS/GSDMD-N signaling pathway

Background: Salidroside (SAL) is the main component of Renus Rhodiola, Studies have shown that it has anti-inflammation, reduces oxidative stress and anti-aging effect. However, its efficacy and mechanism of protecting skin barrier function have not been studied. Purpose: This study demonstrated that whether and how SAL alleviate UVB-induced skin damage and repair skin barrier. Materials and methods: UVB was used to induce photodamage in HaCaT cell line in vitro. MTT assay was used to detect the protective effect of SAL on UVB induced light damage of HaCaT cells, flow cytometry was used to detect the apoptosis and ROS level of HaCaT cells, the related kits were used to detect the relevant indicators of oxidative stress in cells, WB was used to detect protein expression in cells, and AQP3 in HaCaT cell line was overexpressed for further exploration of the way SAL plays its role. BALB/c mice with back hair removed were treated with SAL for 7 days, and the mice were irradiated with UVB every day from the third day. GPSkin was used to detect Trans Epidermal Water Loss (TEWL), hydration and barrier score of mouse skin. HE staining was used to observe the pathological changes of skin lesion, IHC was used to detect the expression of AQP3 and NLRP3, and WB was used to detect the expression of proteins related to pyroptosis. Results: SAL significantly alleviates UVB-induced photodamage by inhibiting the activation of AQP3/ROS/GSDMD-N in damaged skin tissues and keratinocytes, both in vitro and in vivo. This leads to a reduction in skin lesions and levels of IL-18 and IL-1β in cellular supernatants, thereby relieving UVB-induced oxidative stress and downregulation of necroptosis-related proteins (P < 0.05). Conclusion: Thus, our studies demonstrate that SAL can reduce UVB-induced photo damage by down regulating the expression of AQP3 to reduce the transport of H2O2 into keratinocytes. Thus attenuate oxidative stress and NLRP3 dependent cell pyroptosis. Therefore, SAL, could serve as a potential AQP3 inhibitor to alleviate skin photo damage

The MLH1-93 promoter variant influences gene expression

The −93 SNP of MLH1 gene is associated with MLH1 gene methylation in endometrial and colorectal cancers. We undertook luciferase reporter assay and electrophoretic mobility shift assay (EMSA) to test whether the −93 SNP affects the MLH1 gene expression. The luciferase activity for −93A plasmid is significantly lower than −93G plasmid. In EMSA experiments, the −93A and −93G probes have different binding affinity to nuclear proteins of JEG3 cells. Our data indicate that −93 SNP affects MLH1 gene expression by altering protein binding to the promoter of MLH1 gene.The Huazhong University of Science and Technology (24510101 and 25510026

Fu-zi decoction attenuate rheumatoid arthritis in vivo and in vitro by modulating RANK/RANKL signaling pathway

BackgroundFu-zi decoction (FZD) has a long history of application for treating Rheumatoid arthritis (RA) as a classic formulation. However, its underlying mechanisms have not been fully elucidated. This study aimed to decipher the potential mechanism of FZD in treating RA, with a specific focus on receptor activator of nuclear factor κB/receptor activator of nuclear factor κB ligand (RANK/RANKL) signaling pathway.MethodsThe impact of FZD on RA was investigated in collagen-induced arthritis rats (CIA), and the underlying mechanism was investigated in an osteoclast differentiation cell model. In vivo, the antiarthritic effect of FZD at various doses (2.3, 4.6, 9.2 g/kg/day) was evaluated by arthritis index score, paw volume, toe thickness and histopathological examination of inflamed joints. Additionally, the ankle joint tissues were determined with micro-CT and safranin O fast green staining to evaluate synovial hyperplasia and articular cartilage damage. In vitro, osteoclast differentiation and maturation were evaluated by TRAP staining in RANKL-induced bone marrow mononuclear cells. The levels of pro- and anti-inflammatory cytokines as well as RANKL and OPG were evaluated by ELISA kits. In addition, Western blotting was used to investigate the effect of FZD on RANK/RANKL pathway activation both in vivo and in vitro.ResultsFZD significantly diminished the arthritis index score, paw volume, toe thickness and weigh loss in CIA rats, alleviated the pathological joint alterations. Consistent with in vivo results, FZD markedly inhibited RANKL-induced osteoclast differentiation by decreasing osteoclast numbers in a dose-dependent manner. Moreover, FZD decreased the levels of pro-inflammatory cytokines IL-6, IL-1β and TNF-α, while increasing anti-inflammatory cytokine IL-10 level both in serum and culture supernatants. Treatment with FZD significantly reduced serum RANKL levels, increased OPG levels, and decreased the RANKL/OPG ratio. In both in vivo and in vitro settings, FZD downregulated the protein expressions of RANK, RANKL, and c-Fos, while elevating OPG levels, further decreasing the RANKL/OPG ratio.ConclusionIn conclusion, FZD exerts a therapeutic effect in CIA rats by inhibiting RANK/RANKL-mediated osteoclast differentiation, which suggested that FZD is a promising treatment for RA

Exploring the Underlying Mechanisms of Qingxing Granules Treating H1N1 Influenza Based on Network Pharmacology and Experimental Validation

Background: H1N1 is one of the major subtypes of influenza A virus (IAV) that causes seasonal influenza, posing a serious threat to human health. A traditional Chinese medicine combination called Qingxing granules (QX) is utilized clinically to treat epidemic influenza. However, its chemical components are complex, and the potential pharmacological mechanisms are still unknown. Methods: QX’s effective components were gathered from the TCMSP database based on two criteria: drug-likeness (DL ≥ 0.18) and oral bioavailability (OB ≥ 30%). SwissADME was used to predict potential targets of effective components, and Cytoscape was used to create a “Herb-Component-Target” network for QX. In addition, targets associated with H1N1 were gathered from the databases GeneCards, OMIM, and GEO. Targets associated with autophagy were retrieved from the KEGG, HAMdb, and HADb databases. Intersection targets for QX, H1N1 influenza, and autophagy were identified using Venn diagrams. Afterward, key targets were screened using Cytoscape’s protein–protein interaction networks built using the database STRING. Biological functions and signaling pathways of overlapping targets were observed through GO analysis and KEGG enrichment analysis. The main chemical components of QX were determined by high-performance liquid chromatography (HPLC), followed by molecular docking. Finally, the mechanism of QX in treating H1N1 was validated through animal experiments. Results: A total of 786 potential targets and 91 effective components of QX were identified. There were 5420 targets related to H1N1 and 821 autophagy-related targets. The intersection of all targets of QX, H1N1, and autophagy yielded 75 intersecting targets. Ultimately, 10 core targets were selected: BCL2, CASP3, NFKB1, MTOR, JUN, TNF, HSP90AA1, EGFR, HIF1A, and MAPK3. Identification of the main chemical components of QX by HPLC resulted in the separation of seven marker ingredients within 195 min, which are amygdalin, puerarin, baicalin, phillyrin, wogonoside, baicalein, and wogonin. Molecular docking results showed that BCL2, CASP3, NFKB1, and MTOR could bind well with the compounds. In animal studies, QX reduced the degenerative alterations in the lung tissue of H1N1-infected mice by upregulating the expression of p-mTOR/mTOR and p62 and downregulating the expression of LC3, which inhibited autophagy. Conclusions: According to this study’s network pharmacology analysis and experimental confirmation, QX may be able to treat H1N1 infection by regulating autophagy, lowering the expression of LC3, and increasing the expression of p62 and p-mTOR/mTOR