| Rice,as my country's main source of food,produces a large amount of rice bran during its deep processing.Most of the rice bran in our country is used in the feed industry and rarely used in the development of functional active substances.Rice bran contains a large amount of rice bran protein,which is rich in nutrients,low in sensitization,and high in digestibility,and the hydrolyzed active peptide has good biological activity.Therefore,choosing to hydrolyze rice bran protein to obtain active peptides has the advantages of lower cost and higher safety,and can also increase the utilization rate of rice bran,thereby reducing resource waste.This study used natural aging C.elegans,hydrogen peroxide(H2O2)and juglone-induced C.elegans as a model to evaluate the anti-oxidative stress and anti-aging effects of KF-8,a short peptide derived from rice bran,in vivo The effect of anti-oxidation and anti-aging mechanisms have been preliminarily explored.Rice bran active peptide KF-8 is an antioxidant peptide composed of 8 amino acids(KHNRGDEF).Our research group has confirmed that it has a good ability to scavenge free radicals and resist the oxidative stress of HUVEC cells induced by H2O2 in vitro,and proved by this research group to be able to Reduce the oxidative stress and aging process of mice induced by D-galactose in vivo.The purpose of the research of this subject is to further verify the anti-oxidation and anti-aging effects and mechanism of the rice bran active peptide in the body on the basis of the previous research,and to provide a theoretical basis for the development and utilization of rice bran and rice bran protein for human health.And it provides a new way for the value-added development and utilization of the by-products of rice processing.Due to the small size of C.elegans and the short reproductive cycle,it is easy to operate and observe.And more than 65%of its genome is related to humans,so C.elegans is an ideal model in the field of anti-aging research.This topic first selects 0.01 mM,0.03 mM,0.1 mM rice bran active peptides to pre-protect C.elegans for 2 days.Using wild-type naturally aging N2 nematode strains as a model,the rice bran active peptides are used to analyze the natural aging C.elegans life span Impact;analyze the number of eggs laid by the nematode to check the reproductive ability of the naturally senile nematode;record and analyze the number of head and tail swings and the number of body bending in the nematode within 30 s to evaluate the movement ability of the senile nematode;record and analyze the body length of the nematode;Evaluated the activity of antioxidant enzymes(SOD,GSH-px)in senile nematodes and the level of total antioxidant capacity T-AOC;measured the levels of oxidative stress products(ROS,MDA and lipofuscin)in senile nematodes.Finally,this topic selected 0.01 mM,0.03 mM,and 0.1 mM rice bran active peptides to pre-protect C.elegans for 2 days,and analyze the expression levels of related genes:? Using RT-qPCR technology to analyze mRNA expression levels,including genes sod-1,sod-3,sod-5,gst-1,gst-4,gst-10,ctl-1,ctl-2,skn-1,pmk-1,gcs-1,daf-16,mtl-1,akt-1;? Analyze the fluorescence intensity of the reporter gene by using CF1553(sod-3::GFP),CL2166(gst-4::GFP),LD1(skn-1::GFP)and TJ356(daf-16::GFP)are used as model nematodes;? The RNA interference technology was used to interference the skn-1 expression in N2,Using RT-qPCR to detect the expression levels of rice bran active peptides on the N2 strains after skn-1 interference,including genes sod-1,sod-3,gst-1,gst-4,ctl-2,skn-1,pmk-1 and gcs-1,and verify the effect of rice bran active peptides on the ROS level of N2 strain after skn-1 interference.?Using gene-deficient strains EU1(skn-1)and CF1038(daf-16)as models to study the effect of 0.1 mM rice bran active peptide on the lifespan of nematodes.This study found that in aging nematode strains,the pre-protection of 0.1mM rice bran active peptide compared to the control group:? It significantly prolongs the life of the nematode,but does not affect the number of eggs laid,indicating that the peptide is not toxic to the nematode's reproductive ability;?Significantly increase the number of head swings and the frequency of body bending,indicating that the peptide can delay the decline in exercise capacity caused by aging and improve the physiological state of senile nematodes;? Significantly increase the activity of SOD,GSH-px and T-AOC enzymes,Significantly reduced the levels of oxidative stress markers MDA,lipofuscin and ROS.These results preliminarily indicate that the rice bran active peptide has anti-aging and reducing oxidative stress in senile nematodes.In the H2O2 or juglone-induced injury group,the 0.1mM rice bran active peptide pre-protection compared to the group not pre-protected by the rice bran active peptide:?significantly prolonged the life of the nematode;? significantly increased the SOD,GSH-px,T in the nematode body-The enzyme activity of AOC and CAT reduces the levels of MDA,lipofuscin and ROS in the nematode.The above results indicate that the rice bran active peptide can alleviate the oxidative stress of nematodes induced by H2O2 and juglone.During preliminary exploration of the mechanism of action of rice bran active peptides,it was found that the pre-protection of 0.1 mM rice bran active peptides was compared with the blank group:? It significantly increased the expression level of mRNA,including genes sod-1,sod-3,gst-1,gst-4,ctl-2,daf-16,mtl-1,skn-1,pmk-1,gcs-1,while the expression level of akt-1 decreased;?significantly increased CF1553(sod-3::GFP)and The fluorescence intensity of CL2166(gst-4::GFP)and the expression of LD1(skn-1::GFP)promote the nucleation of TJ356(daf-16::GFP);? Compared with N2 which does not interfere with skn-1 expression,in N2(skn-1 RNAi)strain,0.1mM rice bran active peptide group the mRNA expression level of gene gst-4,sod-1,sod-3,ctl-2 and gcs-1 are decreased by 26.4%,25.8%,96.6%,59.6%and 22.24%respectively.In N2(skn-1 RNAi)strain,the level of ROS was reduced by 26%compared to the N2 strain that did not interfere with skn-1 expression.Finally,the lifespan of the skn-1 and daf-16-deficient strains pre-protected with 0.1 mM rice bran active peptide was not significantly different from that of the control group.In summary,rice bran active peptides have antioxidant capacity,which can extend the life of nematodes and improve health indicators.The experiment also shows that skn-1 and daf-16 are essential genes for rice bran active peptides to extend lifespan and resist oxidative stress. |
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