This study aimed to explore the roles of microRNAs (miRNAs) in the post-transcriptional regulation of cocaine- and amphetamine-regulated transcript (CART) peptide in the bovine hypothalamus and to screen key regulatory miRNAs. Targetscan was used to predict the potential miRNAs binding to CART 3' untranslated regions (3'UTR). Bioinformatics analysis predicted 7 miRNA binding sites in the bovine CART 3'UTR, which were bta-miR-377, bta-miR-331-3p, bta-miR-491, bta-miR-493, bta-miR-758, bta-miR-877, and bta-miR-381, respectively. Reverse transcription-PCR (RT-PCR) was carried out to determine the endogenous expression of CART and target miRNAs in the bovine hypothalamus. All the 7 target miRNAs and CART were endogenously expressed in the bovine hypothalamus. The dual-luciferase reporter gene assay was employed to detect the targeted binding relationship between CART 3'UTR and target miRNAs obtained from bioinformatics analysis. The dual-luciferase reporter gene assay confirmed that the 3'UTR of CART had a targeted binding relationship with the 7 target miRNAs. Cell experiments were conducted to examine the effects of target miRNAs on the messenger RNA (mRNA) and protein levels of exogenous CART and screen for key regulatory miRNAs. The results of cell experiments showed that the 7 miRNAs downregulated the mRNA level of CART, with bta-miR-491 demonstrating the strongest downregulating effect. Bta-miR-377, bta-miR-331-3p, bta-miR-491, bta-miR-493, and bta-miR-381 downregulated the protein level of CART, with bta-miR-381 exerting the strongest downregulating effect. Animal experiments were conducted to explore the effects of key regulatory miRNAs on the mRNA and protein levels of CART in the hypothalamus and the CART concentration in the serum. The results from animal experiments showed that miR-491 and miR-381 regulated the endogenous expression of CART in the hypothalamus and the concentration in the serum by binding to the CART 3'UTR. These results suggest that miR-491 and miR-381 are the main miRNAs regulating CART expression in the bovine hypothalamus, which can affect serum CART concentration by modulating endogenous CART expression.
Animals ; MicroRNAs/metabolism* ; Cattle ; Hypothalamus/metabolism* ; 3' Untranslated Regions/genetics* ; Nerve Tissue Proteins/metabolism* ; Gene Expression Regulation ; Binding Sites ; Base Sequence ; Computational Biology/methods* ; Cocaine- and Amphetamine-Regulated Transcript Protein

Oxidative stress, due to the disruption of the balance between reactive oxygen species (ROS) generation and the antioxidant defense system, plays an important role in the pathogenesis of rheumatoid arthritis (RA). Excessive ROS leads to the loss of biological molecules and cellular functions, release of many inflammatory mediators, stimulate the polarization of macrophages, and aggravate the inflammatory response, thus promoting osteoclasts and bone damage. Therefore, foreign antioxidants would effectively treat RA. Herein, ultrasmall iron-quercetin natural coordination nanoparticles (Fe-Qur NCNs) with excellent anti-inflammatory and antioxidant properties were constructed to effectively treat RA. Fe-Qur NCNs obtained by simple mixing retain the inherent ability to remove ROS of quercetin and have a better water-solubility and biocompatibility. In vitro experiments showed that Fe-Qur NCNs could effectively remove excess ROS, avoid cell apoptosis, and inhibit the polarization of inflammatory macrophages by reducing the activation of the nuclear factor-κ-gene binding (NF-κB) pathways. In vivo experiments showed that the swollen joints of mice with rheumatoid arthritis treated with Fe-Qur NCNs significantly improved, with Fe-Qur NCNs largely reducing inflammatory cell infiltration, increasing anti-inflammatory macrophage phenotypes, and thus inhibiting osteoclasts, which led to bone erosion. This study demonstrated that the new metal-natural coordination nanoparticles could be an effective therapeutic agent for the prevention of RA and other diseases associated with oxidative stress.

Objective To analyze the expression of interleukin ( IL)-17A in a mouse model of bleomycin ( BLM)-induced systemic sclerosis ( SSc) and to evaluate its effects on inflammation and fibrosis in skin and lung tissues. Methods Twenty-four female BALB/c mice were randomly divided into four groups:normal control group ( mice were subcutaneously injected with phosphate buffer ) , model group (subcutaneously injected with BLM), antibody group (injected with BLM + IL-17A monoclonal antibody), homotypic control group ( injected with BLM + isotype control) . Pathological changes in skin and lung tis-sues of those mice were observed;inflammatory and fibrotic scores were assessed. Immunohistochemistry and real-time fluorescent quantitative PCR ( RT-PCR) were used to detect the expression of IL-17A, TGF-β1 and typeⅠ collagen in skin and lung tissues of those mice at mRNA level. Mouse lung fibroblasts ( FB) de-rived from the mice of model group were cultured in vitro and then were cultured with IL-17A cytokines with or without the interference of monoclonal antibodies. Expression of typeⅠ collagen and TGF-β1 at mRNA level and levels of IL-6 and TGF-β1 in the culture supernatants were detected by RT-PCR and enzyme-linkedimmunosorbent assay ( ELISA) , respectively. Results Compared with the mice of model and homotypic control groups, those of the antibody group showed mild skin thickening, skin inflammation and lung inflam-mation as well as lower fibrosis scores (P<0. 05). The expression of IL-17A at both protein and mRNA lev-els and the expression of TGF-β1 and collagen typeⅠat mRNA level in skin and lung tissues of mice of the antibody group were significantly lower than those of the model and homotypic control group (P<0. 05). Re-sults of the in vitro cell culture of SSc mice-derived lung FB with IL-17A showed that the expression of TGF-β1 and typeⅠ collagen at mRNA level and the levels of IL-6 and TGF-β1 in the culture supernatants were decreased with the interference of anti-IL-17A monoclonal antibody (P<0. 05), but were still higher than those of the control group (P<0. 05). Conclusion IL-17A promotes the development of inflammation and fibrosis in skin and lung tissues in the mouse model of SSc. Blocking IL-17A might inhibit fibrosis in SSc by inhibiting the production of TGF-β1, IL-6 and typeⅠ collagen.