Plastrum Testudinis Extracts Promote BMSC Proliferation and Osteogenic Differentiation by Regulating Let-7f-5p and the TNFR2/PI3K/AKT Signaling Pathway

Abstract

Background/Aims: Plastrum testudinis extracts (PTE) show osteoprotective effects on glucocorticoid-induced osteoporosis in vivo and in vitro. However, the underlying molecular mechanism of PTE in promoting osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) is unclear. Methods: BMSC proliferation was investigated using the Cell Counting Kit-8 assay. BMSC differentiation and osteogenic mineralization were assayed using alkaline phosphatase and Alizarin red staining, respectively. The mRNA expression levels of Tnfr2, Traf2, Pi3k, Akt, Gsk3β, Runx2, and Ocn were measured using real time quantitative polymerase chain reaction. Protein levels of TNFR2, TRAF2, p-PI3K, p-AKT, p-β-CATENIN, and p-GSK3β were analyzed by western blotting. The functional relationship of Let-7f-5p and Tnfr2 was determined by luciferase reporter assays. Results: The optimum concentration for PTE was 30 μg/ml. PTE significantly promoted BMSC osteogenic differentiation and mineralization after 7 and 14 days in culture, respectively. The combination of PTE and osteogenic induction exhibited significant synergy. PTE upregulated Runx2, and Ocn mRNA expression, and downregulated Tnfr2, Traf2, Pi3k, Akt, and Gsk3β mRNA expression. PTE inhibited TNFR2, TRAF2, and p-β-CATENIN protein expression, and promoted p-PI3K, p-AKT, and p-GSK3β protein expression. In addition, Tnfr2 was a functional target of Let-7f-5p in 293T cells. Conclusions: Our results suggested that PTE may promote BMSC proliferation and osteogenic differentiation via a mechanism associated with the regulation of Let-7f-5p and the TNFR2/PI3K/AKT signaling pathway.

Plastrum Testudinis Extract Mitigates Thiram Toxicity in Broilers via Regulating PI3K/AKT Signaling

“…A recent study on bone MSC proliferation also revealed the upregulation of PI3K, AKT, and GSK3β and downregulation of β-CATENIN and TRAF2 protein expression after the administration of PTE [39]. The possible mechanism of the promotion effect of PTE on the stimulation of osteogenic differentiation is reliant on the Let7f-5p miRNA upregulation and TNFR2/PI3K/AKT signaling pathway [39]. Moreover, the TNFR2 is also considered as the main biomarker for curing cancer [85] osteoclastogenesis, inflammation, and many autoimmune disorders [86].…”

“…Upregulation of p38 MAPK is also associated with an increased amount of immunomodulatory cytokines, such as IL-12 and IL-10 [83], which triggers Th2 and Th1 cell response that protects from infectious agents [84]. A recent study on bone MSC proliferation also revealed the upregulation of PI3K, AKT, and GSK3β and downregulation of β-CATENIN and TRAF2 protein expression after the administration of PTE [39]. The possible mechanism of the promotion effect of PTE on the stimulation of osteogenic differentiation is reliant on the Let7f-5p miRNA upregulation and TNFR2/PI3K/AKT signaling pathway [39].…”

“…Moreover, TD cause lameness which is also an issue regarding animal welfare [13]. The PTE is a mixture of compounds capable of curing osteoporosis and various bone disorders [38,42]; it enhances endochondral ossification by improving the bone regeneration process [39,64].…”

“…A 20 µL, the reaction mixture containing oligo (dT)18, 2× reaction Mix, and 5 µL RNA was used for reverse transcription in a thermocycler (Applied Biosystem CA, USA) at the reaction temperature of 42 • C for 30 min and 85 • C for 5 min [50]. Primer Premier Sofware version 5.0 (Wuhan biotechnology Co. Ltd., Wuhan, China) was used to design the gene specific primers from already reported sequences for respective genes [39] (Table 1). Quantitative RT-PCR was performed using Step One-Plus TM Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) in quadruplex.…”

“…Methyl stearate and methyl palmitate possesses antioxidant properties and provide protection against DPPH-induced oxidation by scavenging free radicals [37]. Plastrum testudinis extract can endorse the multiplication and growth of bone cells and possess significant antioxidant property [38,39].…”