Protective Effects Of Skin By Using EMP During Oxidative Damage And The Study Of Related Mechanisms

Subject: Study extracts of maize plumule( EMP) on the growth effects of skin,and study the effects of EMP on derma structrue and related proteins during oxidative damage. Evaluate the contribution of EMP in anti- oxidation and anti-aging, investigate the related mechanisms, and also explore the growth effects of BMSCs by using EMP.Methods:(1)Mouse derma fibroblast was got by primary cultrue, and the growth effects by using EMP were described by optical microscope, shade selection of MTT and RT-PCR of structure related protein genes. (2)EMP were used in mouse derma fibroblast in advance, and oxidative damage model were made by H2O2, then study the protective effects of fibroblast during the coures of oxidative damage by using the optical microscope, SEM and TEM to observe cells. Hoechst dyeing was used to detect apoptosis ratio, and NADPH distribution in cells also was observed. At last, gene expression ofβ-Actin,Tubulin-α,type I procollagen and ype III procollagen were detected by RT-PCR. (3)After depilation, the mouse skins were painted by EMP ,and 30% H2O2 was used to damage the skin persistently. Then macroscopic observation , optical microscope observation and TEM were used in pathematology experiment. MDA, CAT ,GSH-PX ,MDA and hydroxyproline were also detected to analysis EMP effects in the skin of oxidative damage. (4)EMP were fed to Bombyx mori larvae, then vital movement had been observed to analysis anti-aging effects. The growth situation, abilities of anti-adversity, antioxidase in haemolymph and gene expression of tubulin and collagen in larvae derma were detected, and helped to study the effects of EMP on the body antioxidation system, while the oxidative damage happened in skin partly. (5)Seperated BMSCs from mouse, and added EMP in culture medium to subculture cells. The growth situation, cell cycle and gene expression of two types of procollagen were made as detection index to explore the growth effects of BMSCs by EMP in primary.Results: (1)Add 20 mg/L EMP in culture medium and culture cells for 120 h, OD in MTT can reach 1.097±0.021 and higher than other groups(0.360±0.010～1.023±0.031) (p<0.01).In 96 h,the cell number in single sight were 279.333±15.308,and also higher than 133.000±32.527 in control group(p<0.01). (2)During the course of oxidative damage in vitro, cystoskeleton and subcellular structure in EMP protection group were more integral than the damage group. The apoptosis ratio was (38.38±3.544)% lower than (70.98±3.045)%(p<0.01)damage group. The RT-PCR products'gray scale of Tubulin-α,type I procollagen and ype III procollagen in EMP protection group were 0.811±0.067,0.659±0.026 and 0.549±0.078, higher than 0.628±0.055,0.237±0.035 and 0.217±0.040(p<0.05) respectively. (3)During the course of oxidative damage in the skin in vivo, the structure of skins in EMP protection group and EMP+Vc group were more integral and tight than the damage group. In each test group, the activities of SOD, CAT and GSH-PX were higher than the activities in damage group(p<0.05 or p<0.01). Among the total, the activities of GSH-PX in EMP protection group and EMP+Vc group were 116.793±5.544 and 117.187±4.482 striking higher than 44.515±4.629(p<0.01) in damage group. The contents of hydroxyproline were 23.848±2.721 and 26.970±1.571 in EMP protection group and EMP+Vc group respectively, striking higher than 6.827±1.901(p<0.01),the contents of hydroxyproline in damage group. And the contents of MDA were 10.108±1.836 and 7.258±1.002 respectively in the two test groups. They were both lower than the contents (11.866±1.666) of MDA in damage group(p<0.01) in addition. (4)During the study of aging model of Bombyx mori larvae by EMP, length of lives of Bombyx mori larvae in 20 mg/kg and 40mg/kg EMP feeding groups(617.793±2.4506 h and 23.793±1.378 h) were 4 h and 10 h, longer than the control group(613.997±4.289 h, p<0.01). In addition, abilities of anti-adversity in test group were also better than the control group. Among the skin damage models of Bombyx mori larvae, each test group by using EMP had heavier than the damage group. The EMP protection group which fed 20 mg/kg EMP can lead activities of SOD, CAT raising in haemolymph in larvae body,and activities were 1656.908±108.461 and 26.404±0.068 respectively, higher than 1411.539±79.444 and 17.268±0.136(p<0.01) in damge group. Besides these, EMP also can help protect the expression of collagen gene in larvae derma. (5)In the EMP effects experiment on BMSCs, the proliferation ratio was (61.00±0.72)% in test group higher than (52.73±0.87)%(p<0.01) in control group, and BMSCs also had gene expressions of type I procollagen and type III procollagen.Conclusion: EMP can facilitate proliferation of mouse derma fibroblast, when it was into culture medium. It has the effects of anti-oxidation, and also inhibits the apoptosis happen.EMP can protect cell membrane, cytoskeleton ,other subcellular structures and the expression of procollagen during the oxidative damage. EMP can keep the whole layer skin more integral and thght, and help damage skin recover after oxidative course. Activities of antioxidase can be raised by using EMP in the skin, so it can help clear ROS effectively. EMP also has anti-aging effect. It can help Bombyx mori larvae anti-aging, and promote the body antioxidation system to answer on the part of oxidative damage. In addtion, EMP has interference effect on BMSCs. At the same time, it can facilitate proliferation of BMSCs, and also induce the cell differentiation in some extent.