ABSTRACT
Objective To investigate the effects of cryptotanshinone and tanshinone Ⅱ A,two major monomer components of tanshinone,on the proliferation,differentiation and lipid synthesis of rat meibomian gland epithelial cells in vitro.Methods The eyelid meibomian gland tissue was isolated from 2-month-old SD rats and co-cultured with 3T3 trophoblasts for 5 days.Cryptotanshinone and tanshinone Ⅱ A were prepared with DMSO into different concentrations.The cells were grouped to 0.125 μmol/L,0.250 μmol/L,0.500 μmol/L,1.250 μmol/L and 2.500 μmol/L drug groups and treated for 48 hours,respectively.Only dimethyl sulfoxide (DMSO) was added in medium in the DMSO control group.The expressions of keratin 14 (K14) and p63 in frozen sections of meibomian gland tissue and clones of meibomian gland cells were detected by immunofluorescence.Real-time fluorescence quantitative PCR was used to assay the relative expression of K16,cell proliferation related antigen 67 (Ki67) and CCAAT enhancer binding protein α (C/EBPcα) in meibomian gland cell clones.Crystal violet staining and oil red staining were used to evaluate the colony formation rate and lipid synthesis of meibomian gland epithelial cells.Results Primary cultured meibomian gland cells were cloned on day 4-5 in vivo,and the cloning area was increased on day 7 after culture,p63 and K14 were positively expressed in clones.Compared with the DMSO control group,the relative expression levels of Ki67 mRNA were significantly elevated in the 0.500 μmol/L,1.250 μmol/L and 2.500 μmol/L cryptotanshinone groups (all at P ＜ 0.05).The relative expressions of Ki67 mRNA in the 0.500 μmol/L and 1.250 μmol/L tanshinone Ⅱ A groups were significantly higher than those in the DMSO control group (all at P＜0.05).No significant difference was found in the relative expression of K16 and C/EBPα mRNA among different concentrations of cryptotanshinone or tanshinone Ⅱ A group (all at P＞0.05).No lipid drop was found in the tarsal gland cell clones;however,the accumulation of lipid was seen in the cell clusters at the margin of the clones by oil red O staining.The average clone formation rate of tarsal gland cells in the 1.250 μ mol/L cryptotanshinone group was (2.55±0.20)％,which was significantly higher than (2.05±0.13)％ in the DMSO control group (t =4.379,P＜0.05).The average clone formation rate of tarsal gland cells in the 1.250 μmol/L tanshinone Ⅱ A group was (2.25±0.20)％,there was no significant difference between 1.250 μmol/L tanshinone Ⅱ A group and DMSO control group (t=1.616,P＞0.05).Conclusions Cryptotanshinone and tanshinone Ⅱ A promote the proliferation of meibomian gland epithelia cells,but play less impacts to lipid synthesis of meibomian gland epithelia cells in vitro.cryptotanshinone promote the clone tormation of meibomian gland epithelia cells.