Research Paper Volume 14, Issue 19 pp 7662—7691

Figure 6. Lotus germ extract stimulated collagen production and proliferative ability in aging cells in 3-dimensional gel culture. (A) Young NB1RGB cells, or Aging NB1RGB cells were treated with DMSO (−) or the indicated concentration of lotus germ extract for 72 h and subjected to measurement of collagen content. (B and C) Lotus germ extract stimulated GRP78 expression and protein folding in the ER. (B) Young and aging NB1RGB cells were treated with DMSO (−) or 50 μg/mL lotus germ extract (+) for 24 h and subjected to qPCR. (C) Aging NB1RGB cells expressing SEAP were transduced with a pSEAP2-Control vector and washed 18 h after transduction, and the cells were treated with DMSO (−) or 50 μg/mL lotus germ extract for 24 h. The medium was then changed, and the cells were cultured for another 18 h. Culture media were analyzed for SEAP activity, and luminescence was normalized to cell number. (D–H) Lotus germ extract stimulated collagen production and proliferative ability in 3-dimensional atelocollagen gel culture. Aging NB1RGB cells were treated with DMSO (−) or 50 μg/mL lotus germ extract for 72 h. Equal numbers of these cells and young NB1RGB cells were cultured in atelocollagen gel and treated with or with 50 μg/mL lotus germ extract for 10 days. (D) Representative image showing collagen gel contraction (upper panel). H&E staining of 3-dimensional atelocollagen gel sections. Scar bar: 100 μm (lower panel). (E) Gel surface area. (F) The cell density in a 3-dimensional gel was calculated by using H&E staining. (G) The cell number in the 3-dimensional gel was counted based on degradation of the atelocollagen gel. (H) Extracellular collagen and hyaluronan were measured by immunofluorescence staining. Scar bar: 20 μm. Data are presented as the mean ± SD of three simultaneously performed experiments (A–C, E–G). Each P value was calculated using two-way ANOVA; n.s.: not significant, ^*P < 0.05, ^**P < 0.01.