Uncovering the Key miRNAs and Targets of the Liuwei Dihuang Pill in Diabetic Nephropathy-Related Osteoporosis based on Weighted Gene Co-Expression Network and Network Pharmacology Analysis.

BACKGROUND: Diabetic nephropathy-related osteoporosis (DNOP) is the most common comorbid bone metabolic disorder associated with diabetes mellitus (DM). The Liuwei Dihuang Pill (LWD) is a traditional Chinese herbal medicine widely used to treat diabetic complications, including diabetic nephropathy (DN). This study aimed to identify the biomarkers of the mechanisms of DNOP in LWD with systems biology approaches. METHODS: Herein, we performed an integrated analysis of the GSE51674 and GSE63446 datasets from the GEO database via weighted gene co-expression network and network pharmacology (WGCNA) analysis. In addition, a network pharmacology approach, including bioactive compounds, was used with oral bioavailability (OB) and drug-likeness (DL) evaluation. Next, target prediction, functional enrichment analysis, network analysis, and virtual docking were used to investigate the mechanisms of LWD in DNOP. RESULTS: WGCNA successfully identified 63 DNOP-related miRNAs. Among them, miR-574 was significantly upregulated in DN and OP samples. A total of 117 targets of 22 components associated with LWD in DNOP were obtained. The cellular response to nitrogen compounds, the AGERAGE signaling pathway in diabetic complications, and the MAPK signaling pathway were related to the main targets. Network analysis showed that kaempferol and quercetin were the most significant components. MAPK1 was identified as a potential target of miR-574 and the hub genes in the protein-protein interaction (PPI) network. The docking models demonstrated that kaempferol and quercetin had a strong binding affinity for Asp 167 of MAPK1. CONCLUSION: This study demonstrated that miR-574 may play important roles in DNOP, and the therapeutic effects of kaempferol and quercetin on LWD in DNOP might be mediated by miR-574 by targeting MAPK1. Our results provide new perspectives for further studies on the anti-DNOP mechanism of LWD.

Therapeutic potential of Liuwei Dihuang pill against KDM7A and Wnt/ß-catenin signaling pathway in diabetic nephropathy-related osteoporosis.

The effects of Liuwei Dihuang pill (LWDH) on diabetic nephropathy-related osteoporosis (DNOP) are unclear. The present study aimed to evaluate the effects of LWDH on KDM7A and Wnt/ß-catenin signaling pathway in DNOP rats and the high glucose-induced MC3T3-E1 cells. A DNOP model was prepared by streptozotocin in 9-week-old male Sprague-Dawley (SD) rats to evaluate the effects of LWDH. The cell viability and differentiation capacity of high glucose-induced MC3T3-E1 cells were determined by CCK-8 assay, Alizarin Red staining, and alkaline phosphatase (ALP) staining, respectively. Furthermore, the expressions of KDM7A and Wnt1/ß-catenin pathway-related proteins were determined by Western blot analysis. Treatment of DNOP rats with LWDH could significantly ameliorate the general state, degradation of renal function, and renal pathological changes. LWDH decreased the levels of TNF-α, IL-6, IL-8, IL-1ß, ALP, and TRAP, and increased the calcium, phosphorus in serum, as well as decreased the level of the calcium and phosphorus in the urine. Besides, LWDH significantly improved bone mineral density (BMD), bone volume (BV), and the bone microstructure of DNOP rats. Moreover, LWDH increased the levels of the elastic modulus, ultimate load, and bending strength in the femurs. In MC3T3-E1 cells, serum-containing LWDH significantly increases in cell viability and osteoblastic differentiation capability. The expression of α-SMA, vimentin, KDM7A, Wnt1 and ß-catenin were significantly down-regulated, and the E-cadherin, H3K9-Me2, H3K27-Me2, BMP-4, BMP-7, Runx2, osteocalcin, and Col1a1 were significantly up-regulated with LWDH treatment. The present study shows that LWDH has a therapeutic effect on DNOP, in part, through down-regulation of KDM7A and Wnt/ß-catenin pathway.