MiR-137 promotes TLR4/NF-κB pathway activity through targeting KDM4A, inhibits osteogenic differentiation of human bone marrow mesenchymal stem cells and aggravates osteoporosis.

PURPOSE: As the global population ages rapidly, osteoporotic fractures have become an important public health problem. Previous studies have suggested that miR-137 is involved in the regulation of bone formation, but its specific regulatory mechanism remains unclear. In this study, we aimed to explore the expression, role, and regulatory mechanism of miR-137 in the osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs). METHODS: hBMSCs were induced into osteoblasts at first, and the expression level of miR-137 at different time points was detected. After knockdown and overexpression of miR-137, the effect of miR-137 on the osteogenic differentiation of hBMSCs was examined through alkaline phosphatase (ALP) staining and Alizarin Red staining. Western blotting was performed to detect the expression of runt-related transcription factor 2 (Runx2), osteocalcin (OCN), and toll-like receptor 4 (TLR4)/nuclear factor-κB (NF-κB) pathway. Bioinformatics websites were used to predict the target binding sites for miR-137 and KDM4A, and the results were validated using luciferase reporter gene experiments. Moreover, the ALP activity, calcium nodule formation, and activation of Runx2, OCN, and TLR4/NF-κB pathways were observed after knockdown of KDM4A. RESULTS: The expression of miR-137 decreased during osteogenic differentiation. Knockdown of miR-137 expression increased the osteogenic ability of hBMSCs, while overexpression of it weakened the ability. Through the activation of the TLR4/NF-κB pathway, miR-137 inhibited osteogenic differentiation. KDM4A was identified as a predicted target gene of miR-137. After knocking down KDM4A expression, the osteogenic ability of hBMSCs was diminished, and the TLR4/NF-κB pathway was activated. Furthermore, the osteogenic ability of hBMSCs was partially restored and the activation level of TLR4/NF-κB was reduced after miR-137 knockdown. CONCLUSION: MiR-137 enhances the activity of the TLR4/NF-κB pathway by targeting KDM4A, thereby inhibiting the osteogenic differentiation of hBMSCs and exacerbating osteoporosis.

Pharmacological Inhibition of Macrophage Toll-like Receptor 4/Nuclear Factor-kappa B Alleviates Rhabdomyolysis-induced Acute Kidney Injury / 中华医学杂志(英文版)

<p><b>Background:</b>Acute kidney injury (AKI) is the most common and life-threatening systemic complication of rhabdomyolysis. Inflammation plays an important role in the development of rhabdomyolysis-induced AKI. This study aimed to investigate the kidney model of AKI caused by rhabdomyolysis to verify the role of macrophage Toll-like receptor 4/nuclear factor-kappa B (TLR4/NF-κB) signaling pathway.</p><p><b>Methods:</b>C57BL/6 mice were injected with a 50% glycerin solution at bilateral back limbs to induce rhabdomyolysis, and CLI-095 or pyrrolidine dithiocarbamate (PDTC) was intraperitoneally injected at 0.5 h before molding. Serum creatinine levels, creatine kinase, the expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6, and hematoxylin and eosin stainings of kidney tissues were tested. The infiltration of macrophage, mRNA levels, and protein expression of TLR4 and NF-κB were investigated by immunofluorescence double-staining techniques, reverse transcriptase-quantitative polymerase chain reaction, and Western blotting, respectively. In vitro, macrophage RAW264.7 was stimulated by ferrous myoglobin; the cytokines, TLR4 and NF-κB expressions were also detected.</p><p><b>Results:</b>In an in vivo study, using CLI-095 or PDTC to block TLR4/NF-κB, functional and histologic results showed that the inhibition of TLR4 or NF-κB alleviated glycerol-induced renal damages (P < 0.01). CLI-095 or PDTC administration suppressed proinflammatory cytokine (TNF-α, IL-6, and IL-1β) production and macrophage infiltration into the kidney (P < 0.01). Moreover, in an in vitro study, CLI-095 or PDTC suppressed myoglobin-induced expression of TLR4, NF-κB, and proinflammatory cytokine levels in macrophage RAW264.7 cells (P < 0.01).</p><p><b>Conclusion:</b>The pharmacological inhibition of TLR4/NF-κB exhibited protective effects on rhabdomyolysis-induced AKI by the regulation of proinflammatory cytokine production and macrophage infiltration.</p>

Resolvin D1 Protects Lipopolysaccharide-induced Acute Kidney Injury by Down-regulating Nuclear Factor-kappa B Signal and Inhibiting Apoptosis / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Resolvin D1 (RvD1) is a newly found anti-inflammatory bioactive compound derived from polyunsaturated fatty acids. The current study aimed to explore the protective effect of RvD1 on lipopolysaccharide (LPS)-induced acute kidney injury (AKI) and its possible mechanism.</p><p><b>METHODS</b>Both in vivo and in vitro studies were conducted. Male BALB/c mice were randomly divided into control group (saline), LPS group (LPS 5 mg/kg), RvD1 group (RvD1 5 μg/kg + LPS 5 mg/kg), and blockage group (Boc-MLP 5 μg/kg + RvD1 5 μg/kg + LPS 5 mg/kg). Boc-MLP is a RvD1 receptor blocker. The mice were intraperitoneally injected with these drugs and recorded for general condition for 48 h, while the blood and kidneys were harvested at 2, 6, 12, 24, and 48 h time points, respectively (n = 6 in each group at each time point). Human proximal tubule epithelial cells (HK-2) were randomly divided into control group (medium only), LPS group (LPS 5 μg/ml), RvD1 group (RvD1 10 ng/ml + LPS 5 μg/ml), and blockage group (Boc-MLP 10 ng/ml + RvD1 10 ng/ml + LPS 5 μg/ml). The cells were harvested for RNA at 2, 4, 6, 12, and 24 h time points, respectively (n = 6 in each group at each time point). Blood creatinine was tested by using an Abbott i-STAT portable blood gas analyzer. Tumor necrosis factor-α (TNF-α) level was detected by ELISA. Kidney pathology was observed under hematoxylin and eosin (HE) staining and transmission electron microscope (TEM). We hired immune-histological staining, Western blotting, and fluorescence quantitative polymerase chain reaction to detect the expression of RvD1 receptor ALX, nuclear factor-kappa B (NF-κB) signaling pathway as well as caspase-3. Kidney apoptosis was evaluated by TUNEL staining.</p><p><b>RESULTS</b>RvD1 receptor ALX was detected on renal tubular epithelials. Kaplan-Meier analysis indicated that RvD1 improved 48 h animal survival (80%) compared with LPS group (40%) and RvD1 blockage group (60%), while RvD1 also ameliorated kidney pathological injury in HE staining and TEM scan. After LPS stimulation, the mRNA expression of toll-like receptor 4, myeloid differentiation factor 88, and TNF-α in both mice kidneys and HK-2 cells were all up-regulated, while RvD1 substantially inhibited the up-regulation of these genes. Western blotting showed that the phosphorylated-IκB/IκB ratio in LPS group was significantly higher than that in the control group, which was inhibited in the RvD1 group. RvD1 could inhibit the up-regulation of cleaved-caspase-3 protein stimulated by LPS, which was prohibited in RvD1 blockage group. RvD1 group also had a lower proportion of apoptotic nuclei in mice kidney by TUNEL staining compared with LPS group.</p><p><b>CONCLUSION</b>In LPS-induced AKI, RvD1 could decrease TNF-α level, ameliorate kidney pathological injury, protect kidney function, and improve animal survival by down-regulating NF-κB inflammatory signal as well as inhibiting renal cell apoptosis.</p>