Effects of miR-195 on diabetic nephropathy rats through targeting TLR4 and blocking NF-κB pathway

L.-L. Zhu, H.-Y. Wang, T. Tang

Department of Health, Liaocheng People’s Hospital, Liaocheng, China. tt8646@163.com

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• Metabolism

OBJECTIVE: The aim of this study was to explore the effects of micro ribonucleic acid (miR)-195 on diabetic nephropathy (DN) rats through targeting Toll-like receptor 4 (TLR4) and inhibiting the nuclear factor-κB (NF-κB) signaling pathway.

MATERIALS AND METHODS: The model of DN was first successfully established in rats. All rats were randomly divided into six groups, including control group (n=20), model group (n=20), 25 nM miR-195 mimics group (25 nM M group, n=20), 50 nM M group (n=20), 25 nM miR-195 inhibitor group (25 nM I group, n=20), and 50 nM I group (n=20). Urine volume, proteins and inflammatory factors were detected in each group, respectively. Subsequently, macrophages were cultured and transfected in vitro. The mRNA expressions of miR-195 and TLR4 in control group and model groups were determined using fluorescence quantitative polymerase chain reaction (qPCR). The protein expressions of TLR4 and NF-κB in macrophages were determined using Western blotting. Furthermore, the proliferation of macrophages was detected via cell counting kit-8 (CCK-8) assay.

RESULTS: Compared with model group, 24-h urine volume, urine protein, creatinine, urea nitrogen, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and IL-6 levels declined significantly in 25 nM M group and 50 nM M group (p<0.05). However, they increased significantly in 25 nM I group and 50 nM I group (p<0.05). It could be suggested that miR-195 mimics might relieve the symptoms of DN rats. In kidney tissues in DN, miR-195 was lowly expressed, whereas TLR4 was highly expressed (p<0.01). This suggested that there was a negative correlation between the mRNA expressions of miR-195 and TLR4 (r2=0.4836, p=0.0007). After overexpression of miR-195, the protein expression of TLR4 was significantly reduced (p<0.01), indicating that miR-195 could negatively regulate the protein expression of TLR4. Besides, the protein expressions of TLR4 and NF-κB in si-TLR4 group were evidently lower than those in NC group (p<0.01). Meanwhile, they also had significant differences in si-TLR4 group compared with si-TLR4 + miR-195 inhibitor group (p<0.05). The above results demonstrated that the protein expressions of TLR4 and NF-κB in macrophages could be markedly inhibited by si-TLR4, but be promoted by si-TLR4 + miR-195 inhibitor. CCK-8 assay demonstrated that the proliferation ability of macrophages was remarkably weaker in miR-195 mimics group than NC group (p<0.001). Furthermore, it was also significantly weaker in si-TLR4 + miR-195 inhibitor group than si-TLR4 group (p<0.05).

CONCLUSIONS: MiR-195 reduces the release of inflammatory factors and inhibits the proliferation of macrophages through targeting TLR4 and blocking the NF-κB pathway, thereby alleviating the symptoms of DN rats.

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