| Objective:(1) Immortalized human keratinocyte cell line were cultured and then stimulated by adding substance P in culture medium, in order to build the cell model for study of berberine derivative HB-13's anti-inflammatory mechanism.(2) To define the safe concentration range of HB-13 on HaCaT cell line.(3) To study the effects of HB-13 on secretion of IL-10 and IFN-y of HaCat cell line and further discuss its anti-inflammatory mechanism.Methods:Part one HaCaT Cell were cultured in DMEM/F12 medium supplemented with 10% FBS in a 5% CO2/95% air incubator at 37℃. When cells were totally adherent and in a good condition, two fold dilution of HB-13 from 80 to 2.5μg/mL were made and added to the medium. After 24 hours'incubation, MTT method was applied to determine the safe concentration of HB-13 on HaCaT cell line.Part two HaCaT cell line were cultured and stimulated by Substance P (1×10-8mol/L). The cell culture supernates were collected and contents of IL-10 and IFN-y were quantified by ELISA method to detect the cell model whether or not setup succeed.Part three HaCaT cell line were cultured and stimulated by Substance P, HB-13 with final concentration of 2.5,5 and l0μg/mL were added respectively and continued to culture for 12,24 and 48 hours. The cell culture supernates were collected and contents of IL-10 and IFN-y were quantified by ELISA method. At the same time, cell slides were made and immunocytochemistry was applied to detect the intracellular expression of IL-10 and IFN-y mRNA contents of IL-10 and IFN-y were determined by Real-Time PCR. Triptolide (T0.) was taken as positive control.Experiment results were presented as mean±SD. Statistical analysis was performed using the SPSS 11.5 software package.Results:(1) The results of observing the morphous of HaCaT cultured in vitro showed that cells were round, oval or spindle-type and irregular shape, it was consistent with the reported results of records. (2) While the final concentrations of HB-13 were 2.5,5,10,20,40 and 80μg/mL, the absorbance were 0.982±0.041,0.980±0.043,0.979±0.042,0.728±0.052, 0.564±0.053,0.323±0.053, respectively. Compared with normal cultured cells' absorbance (0.982±0.037), there were no statistic significance when HB-13 at concentration of 2.5,5,10μg/m(p>0.05)L, however, significant difference could be observed when HB-13 at concentration of 20,40,80μg/mL. The results showed that 20,40,80μg/mL HB-13 is harmful on HaCaT cells. In this way, we took 2.5,5,10μg/mL as HB-13's safe concentrations.(3) Substance P was applied to stimulate the HaCaT cells, and the adding of different concentration(2.5,5,1Oμg/mL) of HB-13 in the culture medium for 12,24 and 48 hours further enhanced the secretion of IL-10 which was tested by ELISA method. Compared with the control group which triptolide (T0) rather than HB-13 was added in the medium, the increase of IL-10 was not obvious at 12 and 24 hours when the concentration of HB-13 was 2.5μg/mL. However, as the concentration of HB-13 and culturing time increased, IL-10 was much more higher than To group (p< 0.01), and the difference between groups were statistic significant (p<0.05). We also performed Real-Time PCR and immunocytochemistry to detect the mRNA and protein content of IL-10 in HaCaT cells, the results showed the same tendency as ELISA results displayed.(4) Three different(2.5,5,1Oμg/mL) concentrations of HB-13 at 12,24 and 48 hours time points all showed the ability to increase the secretion of IFN-y. The effect of HB-13 on the secretion of IFN-y was much more significant than that of triptolide (T0), but no difference was found between groups with different concentrations of HB-13 and different time points. The intracellular mRNA and protein content detected by Real-Time PCR and immunocytochemistry is consistent with ELISA results.Conclusion:(1) The safe concentration range of Berberine derivative HB-13 for HaCaT cell line was between 2.5 and 10μg/mL(2) HB-13's anti-inflammatory mechanism is partly due to its enhancement of secretion of cell cytokines IL-10 and IFN-y. |
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