| Royal jelly,secreted from young worker honeybees’ hypopharyngeal and mandibular glands,is the specific food for the queen bee and bee larvae.It is a special pulpous substance which is a little sweet,sour and spicy.Many studies showed that royal jelly was a kind of natural food fit for human health,and had antibacterial,anti-inflammatory,antitumor,hypotensive,promoting tissue growth and other functions.In recent years,studies on royal jelly have mainly focused on antitumor,anticancer,and immunity enhancement effects,while much less research on wound healing has been carried out.In fact,royal jelly has been applied to promote would healing since ancient times.Therefore,this study aimed to isolate protein components from royal jelly and to establish an in vitro wound model to evaluate the effects of royal jelly and its protein components on wound healing,and compare the functional differences of different protein components,and preliminary explore the mechanism of wound healing activity at the cellular level.It will provide theoretical basis for the clinical application of royal jelly on wound healing and provide clues toward the treatment of wound.The main findings are as follows:(1)Effects of royal jelly on the growth of human keratinocytes:The experiment used CCC-ESF-1 cells and HaCaT cells as test cell lines.CCC-ESF-1 cells were found to be insensitive to royal jelly in the tested concentration range without significant difference compared to negative control.Therefore,no further studies were carried out using CCC-ESF-1 cells.When HaCaT cells were cultured with royal jelly,it was found that royal jelly could promote cell growth to some extent,and the effective concentration was 3.9 μg/mL and 7.8 μ/mL at 48 h and 72 h,but higher concentration had an inhibitory effect on cell growth.Subsequently,the evaluation of the effect of royal jelly on promoting cell migration using HaCaT in vitro wound model showed that royal jelly significantly improved the wound healing at the rate of about 42.6%at 256 μg/mL,and the wound healing rate in the negative control group was 35.11%;(2)Isolation of protein components from royal jelly:Referring to the existing literature,we isolated relatively pure MRJP1 from royal jelly by three times of ultracentrifugation.In addition,we also collected the supernatant of royal jelly after being dissolved in water and low-speed centrifugation.It is followed by three days’ dialysis with a 1 kDa dialysis tubing to extract the water-soluble protein components from royal jelly.After freeze-drying,the water-soluble protein components were separated by chromatography with AKTATM pure protein purification system fitted with a superdex 75 gel column.After optimization of the separation conditions,the optimal extraction and separation conditions were:flow rate 0.7 mL/min,sample loading 1 mg,ddH2O elution.As a result,two major fractions of protein components were isolated and purified from royal jelly,which were labeled as peak 1 and peak 2.The retention time of peak 1 was 8-10 min,and the retention time of peak 2 was 17-19 min.In SDS-PAGE gel analysis,the peak 1 was mainly MRJP1 with a molecular weight of 55 kDa,and the peak 2 was a mixed protein fraction with molecular weights of about 50 kDa and about 60 kDa.(3)Effects of royal jelly protein components on the growth of human keratinocytes:When MTT colorimetric assay was performed on HaCaT cells using isolated MRJP1,the results showed that MRJP1 had no growth promoting effect on HaCaT cells,and the similar results were observed when using the peak 1 fraction collected by the protein purification system.However,when the peak 2 protein fraction was used for the experiment,it was found that the peak 2 proteins had a good growth promotion effect on HaCaT,in which the effective concentrations were 0.8 μg/mL and 1.6 μg/mL.Based on the above results,effective components and effective concentrations were screened for the effects on in vitro wound healing models.Using the culture insert plug-in,a uniform wound size model was created in vitro,and peak 2 proteins were applied to the wound site.Peak 2 protein components at the concentration of 1.6μg/mL significantly promoted wound healing and the healing rate was 52.94%,and in the negative control group,the wound healing rate was only 38.76%,indicating that the protein collected from peak 2 played a role in promoting wound healing.(4)Studies on the mechanisms of promoting human keratinocyte growth by royal jelly protein components:Finally,we preliminarily explored the mechanism of action of the protein components on the growth of human keratinocytes from the perspective of cell cycling.The concentrations selected for cell cycling experiment were the effective concentrations in previous research,which were peak 2 proteins at 0.8 p.g/mL and 1.6μg/mL.In terms of the cell ratio in cell cycling,the peak 2 proteins had no effect on the cell cycling process,indicating that its mechanism of promoting cell growth had nothing to do with the cell cycling,but still needs to be studied from other aspects.Quantitative sequencing of the mixed proteins in peak 2 showed that it mainly contained MRJP2(60%),MRJP3(30%),MRJP7(2%)and MRJP1(1%). |
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