Protective Effect Of Different Molecular Weight Silk Fibroin Against Damage Of UVB Irradiation

Objective:To study the effects of different molecular weight silk fibroin proteins on human immortem epidermal cell proliferation,uv protection and UVB protection in rats.Methods:1. The extracted silk fibroin proteins were degraded by alkaline protease under different conditions grouped according to the orthogonal test to obtain the silk fibroin proteins with different molecular weights;2. MTT method was used to determine the optimal dose of UVB in Ha Ca T cells.MTT assay was used to determine the optimal concentration of fibroin on Ha Ca T cell proliferation.3. MTT assay was used to detect the effects of different groups of fibroin proteins on the proliferation of Ha Ca T cells.4. Establish the UVB damage model of Ha Ca T cells.Ha Ca T cells were seeded on the culture plate with a density of 1×10~4/cm~2.When the cell grew to a density of more than 70%(about 72 hours),the culture plate was removed for experimental detection.The radiation dose was 130m J/cm~2(1.97m W/cm~2,66s).5. Effects of different molecular weight groups of fibroin proteins on UVB damage repair in Ha Ca T cellsThe cell experiment was divided into seven groups:Control group,Damage group,SF group,MW1<3000 Da group,MW=3.0×10~4Da group,MW12<3000 Da group,MW=8.0×10~3Da group;The SF group was the silk fibroin group without degradation,and the last four groups were the silk fibroin group with different molecular weights after degradation.(1)MTT method was used to detect the effects of different molecular weight groups of fibroin proteins on UVB damage repair in Ha Ca T cells.(2)Hoechst33342 staining was used to detect the effects of different molecular weight groups of silk fibroin on morphological apoptosis of Ha Ca T cells after UVB damage repair.(3)Flow cytometry was used to detect the effects of different molecular weight groups of silk fibroin on the apoptosis of Ha Ca T cells after UVB damage repair.(4)Effects of different groups of fibroin on glutathione(GSH)and lactate dehydrogenase(LDH)levels in Ha Ca T cells.6. UVB protection of fibroin proteins with different molecular weight groups in ratsThe dorsal skin of 40 SD rats was used as the midline to cut off the surface hair of 4 cm×4 cm and continue feeding for 24 hours.The glabrous skin of the back was divided into four parts(2 cm×2 cm)and randomly divided into eight groups:Control group,Damage group,Base group,SF group,MW1<3000 Da group,MW=3.0×10~4Da group,MW12<3000 Da group,MW=8.0×10~3Da group;The SF group was the silk fibroin group without degradation,the Base group was the base material without fibroin protein group,and the last four groups were the silk fibroin group with different molecular weights after degradation.The Control group continued to be fed without UVB radiation.Damage group SD rats were given 200m J/cm~2(1.97m W/cm~2,102s)UVB radiation directly after intraperitoneal injection of2%pentobarbital sodium(0.2 ml/100 g)under anesthesia.SD rats in the Base group and the drug administration group were anesthetized and fixed on the rat anatomical plate,and each 2 cm×2 cm part of the back was smeared with each group of drugs.After 30 min of drug action,200m J/cm~2(1.97m W/cm~2,102s)dose of UVB radiation was given,and then sacrificed with broken neck after continued feeding for 24 hours.(2)The protective effect of the drug on UVB in rats was observed by HE staining.(3)Effects of different groups of fibroin on glutathione(GSH)and lactate dehydrogenase(LDH)levels in rats.Results:1.Proliferation and uv protection of human immortalized epidermal cells(Ha Ca T cells)by different molecular weight fibroin proteins(1)The optimal dose of UVB to damage Ha Ca T cells was 130 mj/cm~2.(2)The optimal concentration of silk fibroin for Ha Ca T cells proliferation was 1%.(3)Fibroin proteins of different molecular weight all promoted the proliferation activity of Ha Ca T cells.The effect of MW1<3000 Da group is strongest.2.Effects of different molecular weight groups of fibroin proteins on UVB damage repair in Ha Ca T cells(1)Fibroin proteins with different molecular weight can significantly reduce the inhibition of Ha Ca T cell activity by UVB radiation.(2)Fibroin with different molecular weight can significantly inhibit the apoptosis of Ha Ca T cells.(3)Fibroin protein with different molecular weight can increase the decrease of GSH level caused by UVB radiation and decrease the increase of LDH content caused by UVB radiation.3.To study the protective effects of different molecular weight fibroin proteins on UVB in rats(1)HE staining showed that fibroin proteins of different molecular weights could inhibit uvb-induced skin damage in rats.(2)Fibroin protein with different molecular weight can increase the decrease of GSH level caused by UVB radiation and decrease the increase of LDH content caused by UVB radiation.Conclusion:1.Fibroin proteins of different molecular weights have significant ultraviolet protection and epidermal cell proliferation effects.The effect of MW1<3000 Da group is strongest.2.In vitro and in vivo studies have shown that fibroin proteins of different molecular weights may inhibit oxidative stress,inflammatory response and cell apoptosis caused by UVB radiation through their antioxidant activities.3.Silk fibroin protein with different molecular weight has significant anti-photoaging effect on skin.