Biological effect and mechanism of naringin on human degenerative nucleus pulposus-derived mesenchymal stem cell

English Keywords:Nucleus pulposus-derived mesenchymal stem cells  Naringin  Disc degeneration  Apoptosis  PI3K/Akt signaling pathway

English Abstract:

  【Abstract】 Objectives: To investigate the biological effect of naringin on human degenerative nucleus pulposus-derived mesenchymal stem cell(hNPMSC), and to explore the possible mechanism. Methods: The nucleus pulposus tissue of patients with lumbar degenerated disc disease was collected, and hNPMSCs were isolated and cultured in vitro. MSC identification was performed on the isolated cells by observing cell morphology, flow cytometric immunophenotypic assay, and multilineage differentiation. The third generation hNPMSCs were divided into different groups depending on culture medium: control group in normal medium, naringin group in medium with certain concentration of naringin, LY294002 group in medium with certain concentration of naringin and LY294002(PI3K/Akt pathway inhibitor), and the hNPMSCs in each group were incubated for 6 days separately. The apoptosis was detected by flow cytometry and TUNEL staining. The protein expressions of Caspase-3, Bcl-2, Bax and PI3K/Akt signaling pathway-related proteins p-Akt, Akt and p53 were detected by Western Blot. The mRNA expressions of collagen type Ⅱ and aggrecan were detected by RT-PCR. Results: The primary cells showed adherent growth in irregular polygons and transformed into fusiform after passage. The immune phenotype showed that the stem cell-associated positive surface antigen molecules CD73, CD90 and CD105 were highly expressed, but CD45 and CD34 were low expressed. Cells showed osteogenic, chondrogenic and adipogenic differentiation after induced by alizarin red stain, Oil red O stain and toluidine blue stain respectively. Compared with the control group, the apoptosis rate, the rate of TUNEL positive cells, protein expressions of Caspase-3, Bax and p53 in the naringin group were significantly decreased, and the protein expressions of p-Akt and Bcl-2 were significantly increased(P<0.05), but the effect could be reversed by LY294002. The mRNA expressions of collagen type Ⅱ and aggrecan were significantly increased in the naringin group when compared with the control group, but the effect could be weakened by LY294002(P<0.05). Conclusions: Naringin can inhibit apoptosis by activating PI3K/Akt signaling pathway in hNPMSCs and promote differentiation into NP cells.